TW201335185A - Anti-FGFR2 antibodies and uses thereof - Google Patents

Abstract

The present invention provides antibodies, or antigen-binding antibody fragments thereof, or variants thereof which reduce the cell surface expression of FGFR2 after binding to FGFR2 in both cells overexpressing FGFR2 and cells expressing mutated FGFR2. Also provided are antibody-based therapies for FGFR2-related diseases or conditions such as cancer. Antibodies of the invention also can be used in the diagnostics field. The invention also provides nucleic acid sequences encoding the foregoing antibodies, vectors containing the same, pharmaceutical compositions and kits with instructions for use.

Description

anti-FGFR2 antibody and use thereof

The present invention provides recombinant antigen-binding regions and antibodies and functional fragments comprising the antigen-binding regions specific for fibroblast growth factor receptor 2 (FGFR2).

Thus, such antibodies are useful in the treatment of tumors and other conditions and conditions associated with FGFR2 expression. The invention also provides nucleic acid sequences encoding the antibodies, vectors containing the nucleic acid sequences, pharmaceutical compositions, and kits having instructions for use.

Antibody-based therapies have proven to be very effective in treating a variety of cancers, including solid tumors. For example, HERCEPTIN® has been successfully used to treat breast cancer while RITUXAN® is effective against B-cell-associated tumor types. The focus of the development of successful antibody-based therapies is the isolation of antibodies against cell-surface proteins found to be preferentially expressed on tumor cells.

The fibroblast growth factor receptor is tyrosine receptor kinase (RTK), of which four (FGFR1, FGFR2, FGFR3, FGFR4) are known in mammals. 22 human fibroblast growth factor (FGF) was identified as a ligand ((Eswarakumar and Schlessinger, Cytokine & Growth Factor Reviews 2005, 16:139-149; Shimada et al., Proc Natl Acad Sci USA 2001, 98:6500) -6505). FGFR consists of three extracellular immunoglobulin (Ig)-like domains D1-D3 (where domains 2 and 3 are essential for ligand binding), a single transmembrane domain and one containing Urge The cytoplasmic domain of the protein tyrosine kinase core is constructed (see Figure 1 for graphical representation). The extracellular portion additionally has an acid box (AB) and a heparin binding site (HBS) (see Figure 1). An important marker of the FGFR family of RTKs is the presence of variants of diverse alternative splicing. The full-length FGFR2 is called FGFR2α, and the absence of the D1 isoform is called FGFR2β (Fig. 1). Alternative splicing in domain 3 produces two different variants, FGFR2 IIIb with exons 7 and 8, and FGFR2 IIIc with exons 7 and 9 (Fig. 1). The latter splicing affects ligand binding and affects specific patterns. FGFR2 IIIc is mainly expressed by mesenchymal cells, and FGFR2 IIIb is mainly expressed by epithelial cells. FGF7 is also known as keratinocyte growth factor (KGF) and binds only to FGFR2 IIIb, so it is also known as KGFR. Upon binding of FGF to its receptor, FGFR dimerization then proceeds and phosphorylation is cascaded downstream of the FRAS-GRB2 anchored protein complex to the RAS-MAPK signaling cascade with the PI3K-AKT signaling cascade. The first signaling cascade involves cell growth and differentiation, which involves cell survival and fate determination (Katoh and Katoh, Int J Oncol 2006, 29: 163-168).

Careful signaling of all four receptors (FGFR1 to FGFR4) and their splice variants via different FGFs is essential for proper organ formation during embryogenesis (Ornitz et al., Genome Biol 2001, 2:3005). In FGFR2, the absence of all FGFR2 variants caused defects in placenta and limb bud formation and thus caused lethality at E10.5. Specific knockout of FGFR2 IIIb also causes lethality (at P0), which is associated with incomplete development of the lungs, anterior pituitary gland, thyroid, teeth and limbs, whereas FGFR2 IIIc variants Disintegration can survive and shows delayed ossification, proportional short stature, and skull base bony (Eswarakumar and Schlessinger, 2005). Germline activating mutations in FGFR2 in humans cause severe malformations during embryogenesis, such as Abbott's syndrome or Pfeiffer syndrome's coronal and cranial ossification (Robin et al., in Gene Reviews , NCBI Bookshelf Washington, edts. Pagon et al., 1993). In adults, FGFR2 signaling involves wound healing, epidermal repair and cytoprotection in the skin and mucosa (Braun et al., Phil Trans R Soc Lond B 2004, 359: 753-757) and regeneration of damaged liver (Steiling et al) Oncogene 2003, 22: 4380-4388; Böhm, dissertation, Swiss Federal Institute of Technology Zurich, 2009). After infarction, the role of FGFR2 signaling in the migration of epicardial-derived cells (EPDCs) into the heart remains controversial because FGF10/FGFR2 signaling during embryogenesis migrates to EPDCs in dense myocardium (one required for complete cardiac development) Process) is a must (Vega-Hernández et al., Development 2011: 3331-3340; Winter and De Groot, Cell Mol Life Sci 2007, 64: 692-703).

Increased germline-independent signaling through FGFR2 involves different pathologies such as acne (Katoh, J of Invest Dermatol 2009, 129: 1861-1867), psoriasis (Finch et al., Am J Pathol 1997, 151:1619- 1628; Xu et al., J Invest Dermatol 2011: 131: 1521-1529), periodontal disease (Li et al., J Peridontal Res 2005, 40: 128-138), solar plaque (Lin et al., Journal Dermatol Sci 2010, 59: 91-97), Enteropathy (Brauchle et al., J Pathol 1996, 149:521-529), endometriosis (Taniguchi et al., Fertil Steril 2008, 89:478-480), cholesteatoma (Yamamoto-Fukuda et al., Eur Arch Otorhinolaryngol (2008) 265:1173 -1178; d'Alessandro et al., Otol Neurotol. 2010 Sep; 31(7): 1163-9), cholesteatoma chronic otitis media (Yamamoto-Fukuda et al., Otol Neurotol. 2010 Jul; 31(5) : 745-51), atherosclerosis (Che et al., Am J Physiol Heart Circ Physiol 300: H154-H161, 2011) and cancer (see below).

Several studies have publicly emphasized that FGFR2 performance is strongly associated with poor treatment outcomes in cancer patients: Excessive expression of FGFR2 and/or KGF is associated with dilated growth of gastric cancer and shorter patient survival (Matsunobu et al., Int J Cancer 2006, 28: 307-314; Toyokawa et al., Oncol Reports 2009, 21: 875) -880). Therefore, FGFR2 overexpression was detected in 31 to 36.5% of all tested gastric cancer samples (Matsunobu et al., Int J Cancer 2006, 28: 307-314; Toyokawa et al., Oncol Reports 2009, 21: 875- 880). Adenocarcinoma (70% of all gastric cancers) is further divided into two different pathological types, namely intestinal type gastric cancer and diffuse type gastric cancer. Interestingly, the first, less aggressive type is associated with the activation of the ErbB2 oncogenic pathway, which is more aggressive in the FGFR2/PI3K pathway (Yamashita et al., Surg Today 2011, 41:24). -38). About 60% of gastric adenocarcinomas are diffuse and the remaining 40% are of intestinal type (Werner et al., J Cancer Res Clin Oncol 2001, 127: 207-216). FGFR2 overexpression was found in 53% of diffuse gastric cancer samples (Yamashita et al., Surg Today 2011, 41: 24-38). Looking at all the data, HER2 and FGFR2 Performance seems to occur in two different patient populations. The performance of FGFR2 may be partly due to gene amplification, as FGFR2 amplification is found in about 7 to 10% of primary gastric cancer (Kunii et al. Cancer Res 2008, 68: 23-40-2348). In addition, not only FGFR2 expression was found in metastasis, but even more in primary tumors (Yamashita et al., Surg Today 2011, 41: 24-38).

In breast cancer, FGFR2 IIIb expression was found in 57% of tumor samples, but was rare in healthy tissues (Tamaru et al. 2004, 84: 1460-1471). KGF (FGF7) was found in 45% of the samples and was roughly consistent with FGFR2 IIIb. Compared with primary breast cancer that does not express FGF7 or FGFR2 IIIb, the co-expression of FGF7 and its unique receptor FGFR2 IIIb is associated with a significant decrease in the number of apoptotic cells in primary tumors (Tamaru et al. 2004, 84). :1460-1471). Gene amplification was found in gastric cancer and in breast cancer: in 4% of triple negative breast cancer (TNBC) (Turner et al. Oncogene 2010, 29: 2013-2023). Several small nuclear polymorphisms (SNPs) have been identified in breast cancer that are associated with an increased risk of breast cancer (Hunter et al. Nature Genetics 2007, 6: 870-874). If the SNP is in intron 2, it causes a transcriptional upregulation of FGFR2 (Katoh Expert Reviews 2010, 10: 1375-1379). Interestingly, FGFR1 prefers upregulation in ER-positive, whereas FGFR2 prefers upregulation in ER-negative breast cancer (Katoh, Expert Reviews 2010, 10: 1375-1379).

In pancreatic cancer, excessive expression of FGFR2 IIIb and/or FGF7 is strongly associated with venous invasion (Cho et al., Am J Pathol 170: 1964-1974), thereby finding a common expression of FGFR2 and FGF7 in tumor cells, but In and Tumor cells are even more abundant in adjacent stromal cells (Ishiwata et al., Am J Pathol 1998, 153: 213-222).

In epidermal ovarian cancer, 80% of the test cases were found to be up-regulated compared to normal tissues, while 70% FGF7 was present in ascites (Steele et al., Oncogene 20: 5878-5887).

FGFR2 protein was found in all tested aggressive cervical cancers, with strong expression in the aggressive front of tumors (Kawase et al., Int J Oncol 2010, 36:331-340).

In lung adenocarcinoma, FGF7 and FGFR2 were co-expressed in 51.6% of the test cases and correlated with low degree of differentiation, higher proliferation rate, lymph node metastasis, and shorter 5-year survival (Yamayoshi et al., J). Pathol 2004, 204: 110-118).

In endometrial cancer, the most common activating mutation of FGFR2 is found in about 16% of endometrial cancers (Pollock et al., Oncogene 2007, 26: 7158-7162).

In esophageal cancer (EC), FGF7 and FGFR2 are found in cancer cells in 26% of patients with a shorter survival tendency (Yoshino et al., Int J Oncol 2007, 31:721-728).

In hepatocellular carcinoma, FGFR2 is up-regulated by up to 4.7 fold in poorly differentiated tumors. This performance is associated with the incidence of portal vein invasion and lower disease-free survival time (Harimoto et al., Oncology 2010, 78: 361-368).

Several published literatures with experimental in vitro and in vivo data confirm the causal relationship between abnormal FGFR2-signaling and tumor progression:

In the stomach (Takeda et al., Clin Cancer Res 2007; 13: 3051-3057; Kunii et al., Cancer Res 2008; 68: 2340-2348), breast (Turner et al. Oncogene 2010, 29: 2013-2023) ), ovary (Cole et al., Cancer Biol Ther 2010, 10: 495-504) and head and neck squamous cells (Marshall et al., Clin Cancer Res 2011, 17: 5016-5025) cancer cells (Knock- Down) and/or inhibition of FGFR2 results in decreased proliferation of tumor cells and/or increased apoptosis. Also in tumor xenografts, FGFR2 is depleted in FGFR2-expressing tumor cell lines and FGFR2 is inhibited against the stomach (Takeda et al., Clin Cancer Res 2007; 13: 3051-3057) and ovaries (Cole et al., Cancer) Biol Ther 2010, 10: 495-504) cancer cell lines show growth inhibition. In addition, FGF7, which only activates FGFR2, increases gastric cancer in vitro and in vivo (Shin et al., J Cancer Res Clin Oncol 2002, 128: 596-602), breast cancer (Zhang et al., Anticancer Res 1998, 18: 2541). -2546) and proliferation of cancer cell lines in ovarian cancer (Cole et al., Cancer Biol Ther 2010, 10: 495-504). In addition, knockdown of FGFR2 in endometrial cancer cell lines with activated mutant FGFR2 also causes cell cycle arrest and induces cell death (Byron et al., Cancer Res 2008, 68:6902-6907).

FGFR2 signaling promotes gastric cancer cell lines in vitro (Shin et al., J Cancer Res Clin Oncol 2002, 128: 596-602), breast cancer cell lines (Zhang et al., Anticancer Res 1998, 18: 2541-2546) Migration and invasion of pancreatic cancer cell lines (Nomura et al., Br J Cancer 2008, 99: 305-313; Niu et al., J Biol Chem 2007, 282: 6601-6011).

In esophageal cancer, FGFR2 is the most up-regulated gene in tumor-associated fibroblasts. Separation of tumor-associated fibroblasts releases soluble factors that promote esophageal cancer cell proliferation (Zhang et al., hum Cancer Biol 2009, 15: 4017-4022), demonstrating that FGFR2 expressed by stromal cells also promotes tumor cell progression. .

Only a small number of anti-FGFR2 antibodies have been reported. Fortin et al. (J. Neurosci. 2005, 25: 7470-7479) describe a blocking anti-FGFR2 antibody. Wei et al. (Hybridoma 2006, 25: 115-124) showed antibodies specific for FGFR2 IIIb that inhibit KGF-induced cell proliferation. Inhibitory antibodies that bind FGFR2 and FGFR3 are disclosed in WO2007/144893. Antibodies that inhibit FGF binding are disclosed in WO2010/054265 and Zhao et al. (Clin Cancer Res. 2010, 16: 5750-5758). Bai et al. (Cancer Res. 2010, 70:7630-7639) describe antibodies specific for FGFR2 IIIb. R&D Systems sells anti-FGFR2 antibodies that neutralize activity in their assays.

In summary, several FGFR2 splice variants are known. In addition, FGFR2-related diseases are known to be caused by abnormal expression of FGFR2, such as overexpression or amplification of FGFR2, or by various mutant FGFR2 proteins. However, there is a lack of therapies that can cope with many different FGFR2-related diseases.

Summary of invention

The present invention is directed to providing an antibody or antigen-binding antibody fragment thereof which reduces the cell surface expression of FGFR2 after binding to FGFR2 in cells which overexpress FGFR2 and in cells exhibiting mutant FGFR2 Its variant. Also provided are antibody-based therapies against FGFR2-related diseases or conditions, such as cancer, in particular tumors that exhibit FGFR2, such as gastric cancer, breast cancer, pancreatic cancer, colorectal cancer, renal cell carcinoma, prostate cancer, Ovarian cancer, cervical cancer, lung cancer, non-small cell lung cancer (NSCLC), endometrial cancer, esophageal cancer, head and neck cancer, hepatocellular carcinoma, melanoma, and bladder cancer.

The present invention also relates to a polynucleotide encoding the antibody of the present invention or an antigen-binding fragment thereof, a cell expressing the antibody of the present invention or an antigen-binding fragment thereof, a method for producing the antibody of the present invention or an antigen-binding fragment thereof, and the use method The method of inventing an antibody or antigen-binding fragment thereof for inhibiting the growth of dysplastic cells, and the method of using the antibody of the present invention or an antigen-binding fragment thereof for treating and detecting cancer.

The present invention describes antibodies that differ from existing FGFR2 antibodies in that they reduce the surface appearance of FGFR2 in cells overexpressing FGFR2 and in cells expressing mutant FGFR2 upon binding to FGFR2. A specific example of the invention is an antibody or antigen-binding fragment thereof that binds to the extracellular N-terminal epitope of FGFR2 ( ¹ RPSFSLVEDTTLEPE ¹⁵ ) (SEQ ID NO: 63). The antibody of the present invention or antigen-binding fragment thereof will a) activate FGFR2 in a short period of time, b) induce FGFR2 internalization, c) cause efficient decomposition, d) desensitize cancer cells or tumor cells expressing FGFR2, and e) The final result is the anti-tumor activity of these antibodies in in vivo tumor experiments. These and other objects of the invention are more fully described herein.

The antibodies of the invention can be co-administered with known drugs, and in some cases, The antibody itself can be modified. For example, an antibody can bind to a cytotoxic agent, an immunotoxin, a toxic group, or a radioisotope to increase potency more strongly.

The present invention further provides an antibody constituting a tool for diagnosing a malignant or dysplastic condition in which FGFR2 is expressed in comparison to normal tissues or in which FGFR2 is detached from the cell surface and becomes detectable in serum. The prerequisite is that the anti-FGFR2 antibody binds to a detectable label. Preferred labels are radioactive labels, enzymes, chromophores or fluorescent whitening agents.

The present invention also relates to a polynucleotide encoding an antibody of the present invention or an antigen-binding fragment thereof, a cell expressing the antibody of the present invention or an antigen-binding fragment thereof, a method for producing the antibody of the present invention or an antigen-binding fragment thereof, and use The antibody of the present invention or an antigen-binding fragment thereof is used for inhibiting the growth of dysplastic cells, and a method for treating and detecting cancer using the antibody of the present invention or an antigen-binding fragment thereof.

The invention also relates to isolated nucleic acid sequences, each of which encodes an antibody or antigen-binding fragment thereof that is specific for an epitope of FGFR2. The nucleic acids of the invention are suitable for the production of antibodies or antigen-binding antibody fragments in a recombinant manner. Accordingly, the invention also relates to vectors and host cells comprising a nucleic acid sequence of the invention.

The compositions of the invention are useful in therapeutic or prophylactic applications. Accordingly, the invention includes a pharmaceutical composition comprising an antibody of the invention, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier or excipient. In a related aspect, the invention provides a method for treating a condition or condition associated with a cell in which FGFR2 is desired to be present. In a preferred embodiment, The aforementioned condition is cancer. This method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an antibody of the invention as described or contemplated herein.

The invention also provides instructions for using an antibody library to isolate one or more members of the library that specifically bind to FGFR2.

Detailed description of the invention

The present invention is based on the discovery of novel antibodies that have a specific affinity for FGFR2 and that can confer therapeutic benefit to an individual. Antibodies of the invention (which may be human antibodies, humanized antibodies or chimeric antibodies) are useful in a number of respects and will be more fully described herein.

definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. However, the following references may provide a general definition of many of the terms used in the present invention in the art to which the present invention pertains, and such definitions are generally understood and used in the art for reference. Such references include, but are not limited to, Singleton et ah, Dictionary of Microbiology and Molecular Biology (2d ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); Hale & Marham, The Harper Collins Dictionary of Biology (1991); and Lackie et al., The Dictionary of Cell & Molecular Biology (3d ed. 1999); and Cellular and Molecular Immunology, Eds. Abbas, Lichtman and Pober, 2nd Edition, WBSaunders Company. Available in the definition of terms that provide a meaning that is generally understood in the art. There are any other sources of technology used by the average technician. For the purposes of the present invention, the following terms are further defined. Other terms are otherwise defined in the description of the invention. The singular forms "a," "," and "the" Thus, for example, reference to "a gene" refers to one or more genes and includes the same terms as those known to those skilled in the art.

A "human" antibody or antigen-binding fragment thereof is hereby defined as not being chimeric (eg, not "humanized") and not from (all or part of) a non-human species. Thus, a human antibody or antigen-binding fragment thereof can be derived from a human or possibly a synthetic human antibody. "Synthetic human antibody" is defined herein as a sequence having synthetic sequences derived in whole or in part from a computer, based on analysis of known human antibody sequences. In a computer, the design of a human antibody sequence or fragment thereof can be performed, for example, by analyzing a database of human antibody or antibody fragment sequences and designing the polypeptide sequence using the data obtained therefrom. Another embodiment of a human antibody or antigen-binding fragment thereof is encoded by a nucleic acid that isolates a library of antibody sequences of human origin (e.g., based on an antibody library obtained from a human natural source). Examples of human antibodies include antibodies as described in Söderlind et al., Nature Biotech. 2000, 18: 853-856.

"Humanized antibody" or a humanized antigen-binding fragment thereof is defined herein as (i) derived from a non-human source (eg, a gene-transferred mouse bearing a heterologous immune system), the antibody of which is human reproduction Based on the sequence; (ii) the amino acid of the framework region of the non-human antibody is partially replaced by genetic engineering The human amino acid sequence or (iii) the CDR is grafted, wherein the CDRs of the variable domain are from a non-human source, and one or more of the backbone of the variable domain is of human origin and the constant domain, if any, is of human origin .

A "chimeric antibody" or antigen-binding fragment thereof is defined herein as a variable domain derived from a non-human source and some or all of the constant domains being derived from a human source.

The term "monoclonal antibody" as used herein means an antibody obtained from a population of substantially homologous antibodies, ie, a population containing individual antibodies, which is identical except for possible mutations (eg, naturally occurring mutations, which are present in small amounts). ). Thus, the term "single plant" means that the antibody is not a feature of a mixture of isolated antibodies. Relative to a multi-strain antibody population, it typically includes different antibodies directed against different determinants (epitopes), each of which is directed to a single determinant on the anti-antigen. In addition to their specificity, monoclonal antibody preparations are advantageous because they are generally not contaminated by other immunoglobulins. The term "single plant" is not intended to require the production of antibodies by any particular method. The term monoclonal antibody specifically includes chimeric antibodies, humanized antibodies and human antibodies.

As used herein, an antibody "specifically binds to" is an antigen that is "specific" or "specifically recognized" for an antigen of interest, such as a tumor-associated polypeptide antigen (here FGFR2), which binds antigen with sufficient affinity. The antibody can be used as a therapeutic agent in targeting cells or tissues expressing the antigen without significant cross-reactivity with other proteins, or homologous genes and variants (eg, mutant forms, Protein cross-reactors other than splice variants or proteolytic truncated forms). The term "specifically recognizes" or "specifically binds" to an epitope on a particular polypeptide or a particular polypeptide or "specific" to an epitope on a particular polypeptide or a particular polypeptide as used herein, may be an antibody or antigen-binding fragment thereof. Showing, for example, that the antigen has less than about 10 ^-4 M, or less than about 10 ^-5 M, or less than about 10 ^-6 M, or less than about 10 ^-7 M, or less than about 10 ^-8 M, Or a unit price K _{D of} less than about 10 ^-9 M, or less than about 10 ^-10 M, or less than about 10 ^-11 M, or less than about 10 ^-12 M or less. If the antibody is capable of distinguishing between the antigen and one or more reference antigens, the antibody "specifically binds" to the antigen, "specifically" or "specifically recognizes" the antigen. In its most prevalent form, "specific binding", "specific binding", "specific to" or "specific identification" means the ability of an antibody to distinguish between an antigen of interest and an incoherent antigen, such as, for example, according to the following method. One of them was measured. Such methods include, but are not limited to, Western blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans. For example, a standard ELISA assay can be performed. Scoring can be performed according to standard coloration (for example, a secondary antibody with horseradish peroxidase and tetramethylbenzidine with hydrogen peroxide). The reaction in some wells is scored according to optical density, for example at 450 nm. Usually the background (=negative reaction) can be 0.1 OD; usually the positive reaction can be 1 OD. This means that the difference positive/negative is more than 5 times, 10 times, 50 times and preferably more than 100 times. Binding specificity is typically determined by using a set of about three to five incoherent antigens (such as milk powder, BSA, transferrin or the like) that are not a single reference antigen.

"Binding affinity" means the total non-covalent interaction strength of a single binding site of a molecule with its binding partner. Unless otherwise indicated, otherwise defined, as used herein, "binding affinity" means an endogenous binding affinity that reflects a 1:1 interaction between a member of a binding pair (eg, an antibody and an antigen). The dissociation constant "K _D " is often used to indicate the affinity between a molecule (such as an antibody) and its binding partner (such as an antigen), that is, how closely the ligand binds to a particular protein. Ligand-protein affinity is affected by the interaction between two molecules of non-covalent molecules. Affinity can be determined by general methods known in the art, including those described herein. In one embodiment, "K _D " or "K _D value" is determined according to the invention as described in Example 7 by surface plasmon resonance analysis using a Biacore T100 instrument (GE Healthcare Biacore, Inc.). Briefly, antibodies were immobilized on CM5 sensing wafers via an indirect capture reagent (anti-human IgG Fc). Reagents such as "Human Antibody Capture Kit" (BR-1008-39, GE Healthcare Biacore, Inc.) were used as described by the manufacturer. Approximately 5,000 resonance units (RU) of individual mouse anti-human IgG (Fc) antibodies were immobilized per unit. Anti-FGFR2 antibodies were injected to achieve a capture concentration of about 200 to 600 RU. Different concentrations of human, murine, rat, dog and other species-derived FGFR2 peptides containing amino acids 1 to 15 were injected through immobilized anti-FGFR2 antibodies. The sensor map is obtained after the intracellular reference cell correction and then the buffer sample is subtracted. The dissociation equilibrium constant (K _D ) is calculated from the ratio of association (k _on ) to dissociation (k _off ) constant, obtained by fitting the sensorgram to the first-order 1:1 binding model using the Biacore evaluation software. Other suitable devices are BIACORE(R)-2000, BIACORE(R)-3000 (BIAcore, Inc., Piscataway, NJ), or ProteOn XPR36 instrument (Bio-Rad Laboratories, Inc.).

To determine key residues for antibody or antibody fragment binding, epitope fine mapping can be performed using, for example, alanine scanning of the peptide. Thus, each amino acid of the binding epitope was replaced with an alanine residue and the binding of a representative antibody of the invention was tested in an ELISA-based assay. Thus, if the antibody loses more than 50% of its ELISA signal by changing a residue to alanine as described in Example 6, this residue is considered critical for binding.

The term "antibody", as used herein, is intended to mean an immunoglobulin molecule, which preferably contains four polypeptide chains, two heavy (H) chains and two light (L) chains that are normally joined by a disulfide bond. . Each heavy chain contains one heavy chain variable region (abbreviated herein as VH) and one heavy chain constant region. The heavy chain constant region contains, for example, three domains, CH1, CH2, and CH3. Each light chain contains one light chain variable region (abbreviated herein as VL) and one light chain constant region. The light chain constant region contains one domain (CL). The VH and VL regions can be further divided into regions with hypervariability (designated as complementarity determining regions (CDRs)) with a more conserved region (named the framework region (FR)). Each VH and VL consists of three CDRs and up to four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

As used herein, the term "complementarity determining region (CDR; eg CDR1, CDR2 and CDR3) means an amino acid residue of an antibody variable domain, the presence of which is essential for antigen binding. Each variable domain typically has Three CDR regions recognized as CDR1, CDR2 and CDR3. Each complementarity determining region may contain an amino acid residue such as the "complementarity determining region" as defined by Kabat (eg residues in the light chain variable domain of about 24-34 (L1), 50-56 (L2), and 89-97 (L3), and residues 31-35 in the heavy chain variable domain (H1) ), 50-65 (H2) and 95-102 (H3)) (Kabat et al., Sequences of Proteins of Immulological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), And/or those of the "hypermutation loop" (eg, about residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) and heavy chain variable domains in the light chain variable domain 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk; J Mol Biol 196:901-917 (1987)). In some cases, the complementarity determining region may include Amino acids according to the CDR regions defined by Kabat and the hypervariable loop.

Intact antibodies may be assigned to different "classes" depending on the amino acid sequence of their heavy chain constant domain. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into "subclasses" (homotypes), such as IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are referred to as [α], [β], [ε], [γ], and [μ], respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are known. Antibodies as used herein are conventional antibodies and functional fragments thereof.

A "functional fragment" of an antibody/immunoglobulin or an "antigen-binding antibody fragment" is defined herein as a fragment of an antibody/immunoglobulin (eg, a variable region of IgG) that retains an antigen-binding region. The "antigen-binding region" of an antibody is typically in one or more hypervariable regions of the antibody, such as the CDR1, CDR2 and/or CDR3 regions; however, the variable "backbone" region also plays an important role in antigen binding, for example because Provide a skeleton of the CDR. Preferably, the "antigen-binding region" is included to Amino acid residues 4 to 103 of the less variable light (VL) chain and amino acid residues 5 to 109 of the variable heavy (VH) chain, more preferably amino acid residues 3 to 107 and VH of VL Amino acid residues 4 to 111, and particularly preferably intact VL and VH chains (amino acid positions 1 to 109 of VL and amino acid positions 1 to 113 of VH; numbered according to WO 97/08320). A preferred immunoglobulin class for use in the present invention is IgG.

"Functional fragments" or "antigen-binding antibody fragments" of the invention include Fab, Fab', F(ab') ₂ , and Fv fragments; diabodies; single domain antibodies (DAb), linear antibodies; single chain antibody molecules ( scFv); and multispecificity, such as bispecific and trispecific antibodies, formed by antibody fragments (CA K Borrebaeck, editor (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann & S Duebel, editors (2001) Antibody Engineering (Springer Laboratory Manual), Springer Verlag). It will be understood that the binding sites of antibodies other than "multispecific" or "multifunctional" antibodies are each identical. F(ab') ₂ or Fab can be engineered to minimize or completely remove the intermolecular disulfide interaction between the _CH1 and _CL domains.

A variant of an antibody or antigen-binding antibody fragment contemplated in the present invention maintains a binding activity to an antibody or antigen-binding antibody fragment of FGFR2.

Binding proteins contemplated in the present invention are, for example, antibody mimetics, such as Affibodies, Adnectin, Anticalin, DARPin, Avimer, Nano-antibody (Gebauer M. et al. Review, Curr. Opinion In Chem. Biol. 2009; 13: 245-255; Nuttall SD et al., Curr. Opinion in Pharmacology 2008; 8: 608-617).

As used herein, the term "epitope" includes any protein determining position that is capable of specifically binding to an immunoglobulin or T-cell receptor. The epitope determining sites are typically composed of chemically active surface groups of the molecule, such as amino acids or sugar side chains, or combinations thereof, and typically have specific three dimensional structural characteristics, as well as specific charge characteristics. According to any method well known to those skilled in the art, if an antibody appears to compete with a second antibody in a competitive binding assay, the two antibodies are said to "bind to the same epitope."

An "isolated" antibody is one that has been identified and isolated from the cellular components that express it. Contaminant components of a cell are substances that interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other protein or non-protein solutes. In a preferred embodiment, the antibody is purified (1) to greater than 95% by weight of the antibody, such as by, for example, Lloyd's method, UV-Vis spectroscopy or by SDS-capillary gel electrophoresis (eg, at Caliper LabChip GXII, GX) 90 or Biorad Bioanalyzer apparatus), and in preferred embodiments, 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, or (3) to Coomassie blue is used under reducing or non-reducing conditions, or better silver staining is based on the homogeneity of SDS-PAGE. An isolated naturally occurring antibody includes an antibody in situ in a recombinant cell, as at least one component of the natural environment of the antibody is absent. However, isolated antibodies are typically prepared by at least one separation step.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" means a form of cytotoxicity in which binding to Fc is present on certain cytotoxic cells (eg, NK cells, neutrophils, and macrophages). Receptor (FcγR) The secretory Ig is capable of specifically binding these cytotoxic effector cells to the target cells bearing the antigen and thereby killing the target cells, for example using cytotoxins. To assess the ADCC activity of an antibody of interest, an in vitro ADCC assay can be performed, such as described in U.S. Patent No. 5,500,362 or 5,821,337, or U.S. Patent No. 6,737,056 (Presta). Effector cells that can be used for such analysis include PBMC as well as NK cells.

"Complement-dependent cytotoxicity" or "CDC" means the dissolution of a target cell in the presence of complement. Activation of the classical complement pathway begins with the binding of the first complement (Clq) of the complement system to an antibody (of the appropriate subclass) that binds to its cognate antigen. To assess complement activation, a CDC assay can be performed, for example as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). Polypeptide variants (polypeptides having a variant Fc region) having an altered Fc region amino acid sequence and increased or decreased C1q binding are described, for example, in U.S. Patent No. 6,194,551 B1 and WO 1999/51642.

The term immunoconjugate (alternatively meant to be "antibody-drug conjugate" or "ADC") means binding to one or more cytotoxic agents (such as chemotherapeutic agents, drugs, growth inhibitors, toxins (eg, proteins) An antibody to a toxin, a bacterial, fungal, plant-derived enzyme-active toxin or a fragment thereof, or a radioisotope (ie, a radioactive conjugate)). Immunoconjugates have been used for the local delivery of cytotoxic agents (i.e., drugs that kill or inhibit cell growth or proliferation) to treat cancer (e.g., Liu et al., Proc Natl. Acad. Sci. (1996), 93, 8618-8623). The immunoconjugate allows for targeted delivery of the drug moiety to the tumor where it accumulates, wherein systemic administration of the unbound drug may result in unacceptably toxic concentrations of normal cells and/or tissues. antibody Toxins used in toxin conjugates include cytotoxins (such as diphtheria toxin), phytotoxins (such as ricin), and small molecule toxins (such as geldanamycin). These toxins may exhibit their cytotoxic effects by including tubulin binding, DNA binding or topoisomerase inhibition.

"Percent (%) sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as a reference to a multinuclear in a candidate sequence, respectively, after aligning the sequences and introducing intervals (if necessary) to achieve maximum percent sequence identity. The nucleic acid or amino acid residue in the nucleotide or polypeptide sequence is the same percentage of nucleic acid or amino acid residues, respectively. Conservation substitution is not considered part of sequence identity. It is preferably arranged in a non-spaced manner. To determine the arrangement of percent amino acid sequence identity, publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software can be used in different ways, and the techniques of the art are in. Those skilled in the art can determine the appropriate parameters for arranging the sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequence to be aligned.

The term "mature antibody" or "mature antigen-binding fragment", such as a mature Fab variant, includes a derivative of an antibody or antibody fragment that exhibits a more intense binding - that is, a binding with increased affinity - to a particular antigen, such as The extracellular domain of FGFR2. Maturation is the process of identifying a small number of mutations within the six CDRs of an antibody or antibody fragment that increase this affinity. The maturation process is a combination of molecular biological methods that introduce mutations into the antibody and screen to identify improved linkers.

Antibody of the invention

The present invention relates to a method for inhibiting the growth of FGFR2-positive cancer cells and the progression of tumor diseases by providing an anti-FGFR2 antibody. Provided are binding proteins, antibodies, antigen-binding antibody fragments thereof, and variants of antibodies and fragments which are equivalent to cells overexpressing FGFR2 and cells exhibiting mutant FGFR2 which reduce the surface appearance of FGFR2 upon binding to FGFR2. Another embodiment of the present invention provides an antibody or an antigen-binding antibody fragment thereof or a variant thereof, which is conjugated to a widely different cell line exhibiting FGFR2, in cells overexpressing FGFR2 and in cells exhibiting mutant FGFR2, Including but not limited to SNU16 (ATCC-CRL-5974) and MFM223 (ECACC-98050130), which overexpress FGFR2 and AN3-CA (DSMZ-ACC 267) and MFE-296 (ECACC-98031101), which exhibit mutant FGFR2 .

For the purposes of these purposes, a particular embodiment of the invention provides an isolated human, humanized or chimeric antibody, or antigen-binding antibody fragment thereof, which specifically binds to a FGFR2 epitope present in a different form of a mature human FGFR2 polypeptide (eg See SEQ ID NO: 61 of FGFR2α III b and SEQ ID NO: 62 of FGFR2β IIIb, which are expressed by cancer cell lines/cancer cells expressing FGFR2, and/or they are bound by these antibodies with high affinity. As used herein, different "forms" of FGFR2 include, but are not limited to, various isoforms, various splice variants, various glycoforms or FGFR2 polypeptides, which undergo different translational and post-translational modifications. The FGFR2 polypeptide is designated herein as "FGFR2".

Another embodiment of the invention provides antibodies or antigen-binding antibody fragments thereof or variants thereof, which are safe for human administration.

Another embodiment of the invention provides an antibody or antigen-binding antibody fragment thereof or variant thereof that binds to human FGFR2 and cross-reacts with FGFR2 of another species, including but not limited to murine, Rat, rhesus monkey, rabbit, pig and dog FGFR2. Preferably, the other species is a rodent such as, for example, a mouse or a rat. More preferably, the antibodies or antigen-binding antibody fragments thereof or variants thereof bind to human FGFR2 and cross-react with murine FGFR2.

Another embodiment of the invention provides an antibody or antigen-binding antibody fragment thereof or variant thereof, which is effectively internalized upon binding to cells expressing FGFR2. The antibodies of the invention may be co-administered with known drugs, and in some cases the antibodies themselves may be modified. For example, an antibody can bind to a cytotoxic agent, an immunotoxin, a toxic group, or a radioisotope to increase potency more strongly.

Another specific embodiment of the present invention provides an antibody or antigen-binding antibody fragment thereof or a variant thereof which activates FGFR2 in a short period of time and causes FGFR2 to decompose after internalization, thereby allowing different cancer cells or tumor cells expressing FGFR2 to FGF stimulates desensitization and ultimately inhibits tumor growth in vivo.

Another embodiment of the present invention provides an antibody constituting a tool for diagnosing a malignant or dysplastic condition, wherein FGFR2 is elevated compared to normal tissue or wherein FGFR2 is detached from the cell surface and becomes detectable in serum. To. The prerequisite is that the anti-FGFR2 antibody binds to a detectable label. Preferred labels are radioactive labels, enzymes, chromophores or fluorescent whitening agents.

In one aspect, the invention provides an isolated antibody or antigen thereof - A binding fragment comprising an antigen-binding region that binds to FGFR2 expressed by the cell surface and which reduces cell surface expression of FGFR2 in cells overexpressing FGFR2 and cells expressing mutant FGFR2 after binding to FGFR2. In a specific embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof, which comprises FGFR2 which specifically binds to the surface of a naive cell and which binds to FGFR2 in cells exhibiting excessive FGFR2 and cells exhibiting mutant FGFR2 Both antigen-binding regions that reduce the cell surface expression of FGFR2. In one embodiment, the isolated antibody or antigen-binding fragment specifically binds to FGFR2 expressed on the surface of naive cells and binds to at least two different cells that overexpress FGFR2 and at least two mutant FGFR2 after binding to FGFR2 The cell surface appearance of FGFR2 was reduced in different cells.

In still another embodiment, the antibody or antigen-binding fragment thereof specifically binds to FGFR2 expressed on naive, cell surface and (i) in cells overexpressing FGFR2 and cells exhibiting mutant FGFR2 after binding to FGFR2 Decreased cell surface expression of FGFR2, and (ii) caused FGFR2 phosphorylation.

In still another embodiment, the antibody or antigen-binding fragment thereof specifically binds to FGFR2 expressed on the native, cell surface and (i) in cells overexpressing FGFR2 and in cells expressing mutant FGFR2 after binding to FGFR2 Decreasing the cell surface appearance of FGFR2, and (ii) causing FGFR2 phosphorylation, the antibody allows cells expressing FGFR2 to be desensitized using FGF7 stimulation. In yet another embodiment, the desensitization is a cell desensitization that overexpresses FGFR2.

In still another embodiment, the antibody or antigen-binding fragment thereof specifically binds to FGFR2 expressed on the native, cell surface and (i) in cells overexpressing FGFR2 and in cells expressing mutant FGFR2 after binding to FGFR2 Decreased cell surface expression of FGFR2, and (ii) caused internalization of FGFR2, causing FGFR2 to decompose.

In still another embodiment, the antibody or antigen-binding fragment thereof specifically binds to FGFR2 expressed on the native, cell surface and (i) in cells overexpressing FGFR2 and in cells expressing mutant FGFR2 after binding to FGFR2 Decrease the cell surface appearance of FGFR2, and (ii) reduce tumor-growth in allogeneic tumor experiments.

In yet another embodiment, the antibody or antigen-binding fragment thereof is capable of reducing FGFR2 cell surface expression in different cell lines, including, but not limited to, SNU16 (ATCC-CRL-5974) overexpressing FGFR2 MFM223 (ECACC-98050130), and cell line AN3-CA (DSMZ-ACC 267) and mutant MFE-296 (ECACC-98031101) showing mutant FGFR2.

In still another embodiment, the antibody or antigen-binding fragment thereof is capable of overexpressing FGFR2 SNU16 (ATCC-CRL-5974) and MFM223 (ECACC-98050130) after binding to FGFR2, and a cell line exhibiting mutant FGFR2 The surface performance of FGFR2 cells was reduced in AN3-CA (DSMZ-ACC 267) and MFE-296 (ECACC-98031101).

In a preferred embodiment, the cell surface is reduced by at least 10% compared to the surface morphology of the FGFR2 cells of the untreated or control treated cells, 15%, 20%, 25% or 30%.

In yet another preferred embodiment, the cell surface is reduced by at least 10%, 15%, 20%, 25%, or 30% after 96 hours compared to the surface morphology of the FGFR2 cells of the untreated or control treated cells.

In yet another embodiment, the antibody or antigen-binding fragment thereof specifically binds to the extracellular N-terminal epitope of FGFR2 ( ¹ RPSFSLVEDTTLEPE ¹⁵ ) (SEQ ID NO: 63). Key residues for binding to antibodies or antigen-binding fragments thereof that bind to the N-terminal epitope of FGFR2 ( ¹ RPSFSLVEDTTLEPE ¹⁵ ) include, but are not limited to, Arg 1, Pro 2, Phe 4, Ser 5, Leu 6 and Glu 8.

In still another embodiment, the antibody or antigen-binding fragment thereof of the invention binds to an extracellular N-terminal epitope (SEQ ID NO: 63) by at least one epitope residue selected from the group consisting of : Arg 1, Pro 2, Phe 4, Ser 5, Leu 6 and Glu 8.

In still another embodiment, the antibody of the present invention or antigen-binding fragment thereof binds to an extracellular N-terminal epitope (SEQ ID NO: 63) because at least one epitope residue selected from the group consisting of the following residues is Amino acid alanine substitution reduced: Arg 1, Pro 2, Phe 4, Ser 5, Leu 6 and Glu 8.

In still another embodiment, the antibody of the present invention or antigen-binding fragment thereof binds to an extracellular N-terminal epitope (SEQ ID NO: 63) by at least one epitope residue selected from the group consisting of the following residues Media: Pro 2, Leu 6 and Glu 8.

In still another embodiment, the antibody of the present invention or antigen-binding fragment thereof binds to an extracellular N-terminal epitope (SEQ ID NO: 63) because at least one selected from The epitope residues of the group consisting of the following residues are reduced by the substitution of the amino acid alanine: Pro 2, Leu 6 and Glu 8.

In still another embodiment, the antibody of the present invention or antigen-binding fragment thereof binds to an extracellular N-terminal epitope (SEQ ID NO: 63) by at least one epitope residue selected from the group consisting of the following residues The medium: Pro 2, Leu 6 and Glu 8, and the binding to the epitope has no change in the sequence change of position 5 of the epitope.

In still another embodiment, the antibody of the present invention or antigen-binding fragment thereof binds to an extracellular N-terminal epitope (SEQ ID NO: 63) because at least one epitope residue selected from the group consisting of the following residues is Amino acid alanine substitution reduced: Pro 2, Leu 6 and Glu 8, and binding to this epitope did not change for sequence change at position 5 of the epitope.

In still another embodiment, the antibody or antigen-binding fragment thereof loses more than 50% of the ELISA signal by converting at least one amino acid residue of the N-terminal epitope of FGFR2 ( ¹ RPSFSLVEDTTLEPE ¹⁵ ) to alanine. i) the residue is selected from the group consisting of Pro 2, Leu 6 and Glu 8 or (ii) the residue is selected from the group consisting of Arg 1 , Pro 2, Phe 4 and Ser 5 .

In still another embodiment, the isolated antibody or antigen-binding fragment thereof loses more than 50% of the ELISA signal by changing at least one amino acid residue of the N-terminal epitope of FGFR2 ( ¹ RPSFSLVEDTTLEPE ¹⁵ ) to alanine. Wherein the residue is selected from, but not limited to, a) Pro 2, Leu 6 and Glu 8, or b) Arg 1 , Pro 2, Phe 4 and Ser, as shown in Table 7.

In still another embodiment, the antibody or antigen-binding fragment is selected from the group consisting of At least one antibody of the group competes for binding to FGFR2: "M048-D01", "M047-D08", "M017-B02", "M021-H02", "M054-A05", "M054-D03", "TPP -1397", "TPP-1398", "TPP-1399", "TPP-1400", "TPP-1401", "TPP-1402", "TPP-1403", "TPP-1406", "TPP-" 1407", "TPP-1408", "TPP-1409", "TPP-1410", "TPP-1411", "TPP-1412" and "TPP-1415".

In this document, reference is made to the preferred antibodies of the invention shown in Tables 9 and 10 below: "M017-B02", "M021-H02", "M047-D08", "M048-D01", "M054-A05" , "M054-D03", "TPP-1397", "TPP-1398", "TPP-1399", "TPP-1400", "TPP-1401", "TPP-1402", "TPP-1403"," TPP-1406", "TPP-1407", "TPP-1408", "TPP-1409", "TPP-1410", "TPP-1411", "TPP-1412" and "TPP-1415".

M017-B02 represents a variable heavy chain region corresponding to SEQ ID NO: 3 (DNA) / SEQ ID NO: 1 (protein) and a corresponding to SEQ ID NO: 4 (DNA) / SEQ ID NO: 2 ( An antibody to the variable light chain region of a protein).

M021-H02 represents a variable heavy chain region corresponding to SEQ ID NO: 13 (DNA) / SEQ ID NO: 11 (protein) and a corresponding to SEQ ID NO: 14 (DNA) / SEQ ID NO: 12 ( An antibody to the variable light chain region of a protein).

M047-D08 represents a variable heavy chain region corresponding to SEQ ID NO: 23 (DNA) / SEQ ID NO: 21 (protein) and a pair An antibody that is in the variable light chain region of SEQ ID NO: 24 (DNA) / SEQ ID NO: 22 (protein).

M048-D01 represents a variable heavy chain region corresponding to SEQ ID NO: 33 (DNA) / SEQ ID NO: 31 (protein) and a corresponding to SEQ ID NO: (DNA) 34 / SEQ ID NO: 32 ( An antibody to the variable light chain region of a protein).

M054-D03 represents a variable heavy chain region corresponding to SEQ ID NO: 43 (DNA) / SEQ ID NO: 41 (protein) and a corresponding to SEQ ID NO: 44 (DNA) / SEQ ID NO: 42 ( An antibody to the variable light chain region of a protein).

M054-A05 represents a variable heavy chain region corresponding to SEQ ID NO: 53 (DNA) / SEQ ID NO: 51 (protein) and a corresponding to SEQ ID NO: 54 (DNA) / SEQ ID NO: 52 ( An antibody to the variable light chain region of a protein).

TPP-1397 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 83 (protein) and a variable light chain region corresponding to SEQ ID NO: 84 (protein).

TPP-1398 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 93 (protein) and a variable light chain region corresponding to SEQ ID NO: 94 (protein).

TPP-1399 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 103 (protein) and a variable light chain region corresponding to SEQ ID NO: 104 (protein).

TPP-1400 indicates that it contains a corresponding SEQ ID NO: 113 (protein) A variable heavy chain region and an antibody corresponding to the variable light chain region of SEQ ID NO: 114 (protein).

TPP-1401 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 123 (protein) and a variable light chain region corresponding to SEQ ID NO: 124 (protein).

TPP-1402 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 133 (protein) and a variable light chain region corresponding to SEQ ID NO: 134 (protein).

TPP-1403 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 73 (protein) and a variable light chain region corresponding to SEQ ID NO: 74 (protein).

TPP-1406 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 153 (protein) and a variable light chain region corresponding to SEQ ID NO: 154 (protein).

TPP-1407 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 163 (protein) and a variable light chain region corresponding to SEQ ID NO: 164 (protein).

TPP-1408 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 173 (protein) and a variable light chain region corresponding to SEQ ID NO: 174 (protein).

TPP-1409 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 183 (protein) and a variable light chain region corresponding to SEQ ID NO: 184 (protein).

TPP-1410 indicates that it contains a corresponding SEQ ID NO: 193 (protein) A variable heavy chain region and an antibody corresponding to the variable light chain region of SEQ ID NO: 194 (protein).

TPP-1411 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 203 (protein) and a variable light chain region corresponding to SEQ ID NO: 204 (protein).

TPP-1412 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 213 (protein) and a variable light chain region corresponding to SEQ ID NO: 214 (protein).

TPP-1415 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 143 (protein) and a variable light chain region corresponding to SEQ ID NO: 144 (protein). In still another preferred embodiment, the antibody or antigen-binding fragment comprises a heavy or light chain CDR sequence that is at least 50%, 55%, 60%, 70%, 80%, 90 or 95% identical to at least One, preferably corresponding to the CDR sequences of the following antibodies: "M048-D01", "M047-D08", "M017-B02", "M021-H02", "M054-A05", "M054-D03", "TPP- 1397", "TPP-1398", "TPP-1399", "TPP-1400", "TPP-1401", "TPP-1402", "TPP-1403", "TPP-1406", "TPP-1407" , "TPP-1408", "TPP-1409", "TPP-1410", "TPP-1411", "TPP-1412" or "TPP-1415", or at least 50%, 60%, 70%, 80% , 90%, 92% or 95% identical to the VH or VL sequence of the following antibodies: "M048-D01", "M047-D08", "M017-B02", "M021-H02", "M054-A05"," M054-D03", "TPP-1397", "TPP-1398", "TPP-1399", "TPP-1400", "TPP-1401", "TPP-1402", "TPP-1403", "TPP-" 1406", "TPP- 1407", "TPP-1408", "TPP-1409", "TPP-1410", "TPP-1411", "TPP-1412" or "TPP-1415".

In a preferred embodiment, the antibody or antigen-binding fragment of the invention comprises at least one CDR sequence or at least one variable heavy or light chain sequence as set forth in Tables 9 and 10.

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 5 (H-CDR1), SEQ ID NO: 6 (H-CDR2) and SEQ ID NO: 7 (H-CDR3); and a light chain antigen-binding region comprising SEQ ID NO: 8 (L-CDR1), SEQ ID NO: 9 (L-CDR2), and SEQ ID NO: 10 ( L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 15 (H-CDR1), SEQ ID NO: 16 (H-CDR2) and SEQ ID NO: 17 (H-CDR3); and a light chain antigen-binding region comprising SEQ ID NO: 18 (L-CDR1), SEQ ID NO: 19 (L-CDR2), and SEQ ID NO: 20 ( L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 25 (H-CDR1), SEQ ID NO: 26 (H-CDR2) and SEQ ID NO: 27 (H-CDR3); and a light chain antigen-binding region comprising SEQ ID NO: 28 (L-CDR1), SEQ ID NO: 29 (L-CDR2), and SEQ ID NO: 30 ( L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 35 (H-CDR1), SEQ ID NO: 36 (H-CDR2) and SEQ ID NO: 37 (H-CDR3): and comprising a light chain antigen - A binding region comprising SEQ ID NO: 38 (L-CDR1), SEQ ID NO: 39 (L-CDR2), and SEQ ID NO: 40 (L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 45 (H-CDR1), SEQ ID NO: 46 (H-CDR2) and SEQ ID NO: 47 (H-CDR3); and a light chain antigen-binding region comprising SEQ ID NO: 48 (L-CDR1), SEQ ID NO: 49 (L-CDR2) and SEQ ID NO: 50 ( L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 55 (H-CDR1), SEQ ID NO: 56 (H-CDR2) and SEQ ID NO: 57 (H-CDR3); and a light chain antigen-binding region comprising SEQ ID NO: 58 (L-CDR1), SEQ ID NO: 59 (L-CDR2), and SEQ ID NO: 60 ( L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 75 (H-CDR1), SEQ ID NO: 76 (H-CDR2) and SEQ ID NO: 77 (H-CDR3); and a light chain antigen-binding region comprising SEQ ID NO: 78 (L-CDR1), SEQ ID NO: 79 (L-CDR2), and SEQ ID NO: 80 ( L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 85 (H-CDR1), SEQ ID NO: 86 (H-CDR2) and SEQ ID NO: 87 (H-CDR3); and a light chain antigen- A binding region comprising SEQ ID NO: 88 (L-CDR1), SEQ ID NO: 89 (L-CDR2), and SEQ ID NO: 90 (L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 95 (H-CDR1), SEQ ID NO: 96 (H-CDR2) and SEQ ID NO: 97 (H-CDR3); and a light chain antigen-binding region comprising SEQ ID NO: 98 (L-CDR1), SEQ ID NO: 99 (L-CDR2), and SEQ ID NO: 100 ( L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 105 (H-CDR1), SEQ ID NO: 106 (H-CDR2) and SEQ ID NO: 107 (H-CDR3); and a light chain antigen-binding region comprising SEQ ID NO: 108 (L-CDR1), SEQ ID NO: 109 (L-CDR2), and SEQ ID NO: 110 ( L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 115 (H-CDR1), SEQ ID NO: 116 (H-CDR2) and SEQ ID NO: 117 (H-CDR3); and a light chain antigen-binding region comprising SEQ ID NO: 118 (L-CDR1), SEQ ID NO: 119 (L-CDR2), and SEQ ID NO: 120 ( L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 125 (H-CDR1), SEQ ID NO: 126 (H-CDR2) and SEQ ID NO: 127 (H-CDR3); and a light chain antigen- A binding region comprising SEQ ID NO: 128 (L-CDR1), SEQ ID NO: 129 (L-CDR2), and SEQ ID NO: 130 (L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 135 (H-CDR1), SEQ ID NO: 136 (H-CDR2) and SEQ ID NO: 137 (H-CDR3); and a light chain antigen-binding region comprising SEQ ID NO: 138 (L-CDR1), SEQ ID NO: 139 (L-CDR2), and SEQ ID NO: 140 ( L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 145 (H-CDR1), SEQ ID NO: 146 (H-CDR2) and SEQ ID NO: 147 (H-CDR3); and a light chain antigen-binding region comprising SEQ ID NO: 148 (L-CDR1), SEQ ID NO: 149 (L-CDR2), and SEQ ID NO: 150 ( L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 155 (H-CDR1), SEQ ID NO: 156 (H-CDR2) and SEQ ID NO: 157 (H-CDR3); and a light chain antigen-binding region comprising SEQ ID NO: 158 (L-CDR1), SEQ ID NO: 159 (L-CDR2), and SEQ ID NO; L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 165 (H-CDR1), SEQ ID NO: 166 (H-CDR2) and SEQ ID NO: 167 (H-CDR3); and a light chain antigen- A binding region comprising SEQ ID NO: 168 (L-CDR1), SEQ ID NO: 169 (L-CDR2), and SEQ ID NO: 170 (L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 175 (H-CDR1), SEQ ID NO: 176 (H-CDR2) and SEQ ID NO: 177 (H-CDR3); and a light chain antigen-binding region comprising SEQ ID NO: 178 (L-CDR1), SEQ ID NO: 179 (L-CDR2), and SEQ ID NO: 180 ( L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 185 (H-CDR1), SEQ ID NO: 186 (H-CDR2) and SEQ ID NO: 187 (H-CDR3); and a light chain antigen-binding region comprising SEQ ID NO: 188 (L-CDR1), SEQ ID NO: 189 (L-CDR2), and SEQ ID NO: 190 ( L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 195 (H-CDR1), SEQ ID NO: 196 (H-CDR2) and SEQ ID NO: 197 (H-CDR3); and a light chain antigen-binding region comprising SEQ ID NO: 198 (L-CDR1), SEQ ID NO: 199 (L-CDR2), and SEQ ID NO: 200 ( L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 205 (H-CDR1), SEQ ID NO: 206 (H-CDR2) and SEQ ID NO: 207 (H-CDR3); and a light chain antigen- A binding region comprising SEQ ID NO: 208 (L-CDR1), SEQ ID NO: 209 (L-CDR2), and SEQ ID NO: 210 (L-CDR3).

In a preferred embodiment, the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region comprising SEQ ID NO: 215 (H-CDR1), SEQ ID NO: 216 (H-CDR2) and SEQ ID NO: 217 (H-CDR3); and a light chain antigen-binding region comprising SEQ ID NO: 218 (L-CDR1), SEQ ID NO: 219 (L-CDR2) and SEQ ID NO: 220 ( L-CDR3).

For example, an antibody of the invention may be IgG (e.g., IgGl, IgG2, IgG3, IgG4), and the antibody fragment may be Fab, Fab', F(ab')2 or scFv. Accordingly, an antibody fragment of the invention can be or can contain an antigen-binding region that functions in one or more ways as described herein.

For example, the antibody Fab fragment M048-D01 (the VH chain of SEQ ID NO: 31, and the VL chain of SEQ ID NO: 32) is expressed as human IgG1 M048-D01-hIgG1 (the heavy chain of SEQ ID NO: 67, and The light chain of SEQ ID NO: 68, and the Fab fragment M047-D08 (the VH chain of SEQ ID NO: 21, and the VL chain of SEQ ID NO: 22) are expressed as human IgG1 M047-D08-hIgG1 (SEQ ID NO: The heavy chain of 69, and the light chain of SEQ ID NO: 70). For efficient selection, the N-terminal 3 amino acids [EVQ] of the heavy chain (SEQ ID NO: 67 and SEQ ID NO: 69) may also be additionally expressed as [QVE], such as, for example, human IgG1. M048-D01-hIgG1 A variant of the heavy chain (SEQ ID NO: 222). For efficient selection, the N-terminus of the light chain can be extended by an amino acid residue such as alanine.

In a preferred embodiment, the antibody or antigen-binding antibody fragment of the invention is monoclonal. In a more preferred embodiment, the antibody or antigen-binding antibody fragment of the invention is a human, humanized or chimeric antibody or antigen-binding antibody fragment.

In another aspect, the invention provides an antibody or antigen-binding fragment having an antigen-binding region that specifically binds to FGFR2 and/or has a high affinity for FGFR2, the FGFR2 is independent of the alpha and beta isoforms and IIIb Spliced to IIIc (see, for example, FGFR2α IIIb of SEQ ID NO: 61 and FGFR2β IIIb of SEQ ID NO: 62).

An antibody or antigen-binding fragment is said to have "high affinity" for an antigen if the affinity measurement is below 250 nM (the monovalent affinity of the antibody or antigen-binding fragment). Preferably, the antibody or antigen-binding region of the invention binds to human FGFR2 with an affinity of less than 250 nM, preferably less than 150 nM, as determined by the monovalent affinity for human FGFR2. For example, the affinity of an antibody of the invention for FGFR2 of different species may be about 100 nM (monovalent affinity of an antibody or antigen-binding fragment), as exemplified in Table 8 for M048-D1 and M047-D08.

The IgGl format was used in cell-based affinity assays by fluorescence activated cell sorting (FACS). Summarized in Table 6 provides a representative antibody for anti -FGFR2-IgG human origin (SNU16, MFM223), murine source (4T1) binding strength of rat origin (RUCA) cancer cell lines (EC ₅₀₎ of.

If the affinity measured by FACS is less than 100 nM (apparent affinity of IgG), then IgG1 is said to have "high affinity" for an antigen. Preferably, the bivalent antibody or antigen-binding fragment of the invention binds to FGFR2 with an affinity of less than 100 nM, more preferably less than 50 nM, and still preferably preferably less than 10 nM. More preferably, the bivalent antibody Affinity of less than 5 nM, and more preferably less than 1 nM, binds to FGFR2 as determined by the apparent affinity of IgG for FGFR2. For example, the antibody against FGFR2 of the invention differs in human, murine and rat origins. The apparent affinity of the tumor cell line can be about 89.5 nM or less than 0.1 nM as determined by FACS analysis as shown in Table 6.

When the antibody or antigen-binding fragment of the present invention enters the tumor cell expressing FGFR2 for a half maximum internalization time (t1⁄2) of less than 180 minutes or more preferably less than 120 minutes, and more preferably less than 90 minutes, it is efficiently Internalization. More preferably, the antibody or antigen-binding fragment having a half maximum internalization time (t1⁄2) of 60 minutes or less is determined according to the procedure as described in Example 12.

Co-staining of small G-proteins can be used to assess in more detail the carrier path of the antibody after internalization. For example, Rab GTPase, which regulates several steps of membrane trafficking, including vesicle formation, vesicle migration along actin and tubulin networks, and membrane fusion, can be used to distinguish between different pathways. Thus, co-staining of labeled antibodies using Rab7, which is expressed in late phagosomes and lysosomes, indicates that the complex enters the phagosome-lysosomal pathway after internalization of FGFR2, whereas Rab11 is used (in the early days) Co-staining of the performance in the recirculating phagosomes indicates that these antibodies internalize and bind to the recirculation path after binding to FGFR2. Entry into the phagosome-solute pathway allows the antibody to internalize and induce FGFR2 breakdown, which ultimately leads to desensitization of this pathway. Figure 7 shows the co-staining of a representative antibody of the invention using Rab7 and Rab11 as described in Example 12. Type.

The internalizable antibody or antigen-binding fragment of the invention is suitable as a targeted portion of an antibody-drug conjugate (ADC). The antibody or antigen-binding fragment is suitable for use in an in vitro or in vivo method to deliver a compound, preferably a cytotoxic agent, to a cell that exhibits FGFR2.

In some embodiments, an antibody, an antigen-binding fragment thereof or a derivative thereof, or a nucleic acid encoding the same is isolated. An isolated biological component (such as a nucleic acid molecule or a protein such as an antibody) is another biological component that has substantially been derived from a biological cell in which the components naturally occur (eg, other chromosomal or extrachromosomal DNA and RNA, proteins, and cells) Is separated or purified. Nucleic acids and proteins that have been "isolated" include, for example, by Sambrook et al., 1989 (Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring. Nucleic acids and proteins purified by standard purification methods described in Harbor, USA) and Robert K. Scopes et al. 1994 (Protein Purification, - Principles and Practice, Springer Science and Business Media LLC). The term also encompasses nucleic acids and proteins prepared by recombinantly expressing and chemically synthesized nucleic acids in a host cell.

The antibodies of the invention can be derived from a library of recombinant antibodies based on amino acid sequences isolated from antibodies of a large number of healthy volunteers. The complete human CDRs are recombined into new antibody molecules using the n-CoDeR® technology. The unique recombination program allows the library to contain antibodies that are more diverse and diverse than the natural creators of the human immune system.

Antibody production

The intact human N-CoDeR antibody phage display library was used to isolate the FGFR2-specific, human monoclonal antibodies of the invention by combining whole cell and protein panning and by developing specific methods. Such methods include the development of a ability to identify a preferred binding to display on the cell surface, and cross-reactivity with murine FGFR2 and FGFR2 from other species, and have been found to be independent of FGFR2 overexpression and FGFR2-related diseases (such as cancer). Panning of FGFR2 mutations and panning procedures for functionally active antibodies and screening assays.

Antibodies against cell surface FGFR2 were developed in phage-display technology (PDT) by combining three non-conventional methods. First, a number of recombinant, soluble, human and murine FGFR2 Fc-fusion proteins of splice variants (α, β, IIIb and IIIc) were screened to screen for a wide range of splice variant cross-reactivity. Next, cell-surface screening was additionally performed using KATO III cells expressing FGFR2 on their cell surface. Third, development of screening methods that allow for the successful screening of phage products obtained by panning recombinant, soluble, human and murine FGFR1, FGFR2, FGFR3 and FGFR4 Fc fusion proteins of all KATOIII cells and several splice variants. Specific binders (not bound to FGFR1, FGFR3 and FGFR4) with very broad splice variant cross-reactivity were screened against FGFR2.

After identifying the preferred Fab fragment, these are expressed as full length IgG. For example, antibody fragment M048-D01 (the VH chain of SEQ ID NO: 31, and the VL chain of SEQ ID NO: 32) is expressed as human IgG1 M048-D01-hIgG1 (the heavy chain of SEQ ID NO: 67, and SEQ ID NO: The light chain of 68), while the Fab fragment M047-D08 (the VH chain of SEQ ID NO: 21, and the VL chain of SEQ ID NO: 22) is expressed as human IgG1 M047-D08-hIgG1 (heavy chain of SEQ ID NO: 69) And the light chain of SEQ ID NO: 70). For efficient selection, the N-terminal 3 amino acids [EVQ] of the heavy chain (SEQ ID NO: 67 and SEQ ID NO: 69) may also be additionally expressed as [QVE], such as, for example, human IgG1. A variant of the heavy chain of M048-D01-hIgG1 (SEQ ID NO: 222). For efficient selection, the N-terminus of the light chain can be extended by an amino acid residue such as alanine. Such constructs may, for example, be temporarily expressed in mammalian cells as described in Tom et al., Chapter 12 in Methods Express: Expression Systems edited by Micheal R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007. Briefly, CMV-promoter-based expression plastids were transfected into HEK293-6E cells and grown in Fernbach-Flask or shake flasks. It was expressed in F17 medium (Invitrogen) for 5 to 6 days at 37 °C. 5 g/l pancreatic TN1 (Organotechnie), 1% ultra low IgG FCS (Invitrogen) and 0.5 mM valproic acid (Sigma) were supplemented 24 hours after transfection.

The characterization of these antibodies was further carried out in accordance with the binding affinities of these antibodies in ELISA and by BIAcore binding to soluble FGFR2. FACS binding to cells of different species was performed to screen for cell-binding antibodies with high affinity for mouse, rat, and human cancer cell lines.

The combination of these specific methods allows the isolation of the unique antibodies "M017-B02", "M021-H02", "M047-D08", "M048-D01", "M054-A05" and "M054-D03".

Further characterization revealed that the selected antibody binds to a unique epitope at the N-terminus of FGFR2 because of its specific characteristics. These unique antibodies were further characterized in in vitro phosphorylation assays, internalization assays, and in vivo tumor xenograft experiments. Selected antibodies showed strong and significant anti-tumor activity in tumor xenograft experiments using SNU16 cells.

Peptide variant

The antibodies or antigen-binding fragments of the invention are not limited to the particular peptide sequences provided herein. More specifically, the invention also encompasses variants of such polypeptides. With reference to the present disclosure and the available techniques and references, those skilled in the art will be able to prepare, test, and use functional variants of the antibodies disclosed herein, while recognizing that such variants capable of binding to FGFR2 fall within the scope of the present invention Inside.

Variants can include, for example, at least one complementarity determining region (CDR) (high variation) and/or backbone (FR) (variable) domain/position altered as compared to the peptide sequences disclosed herein. To better illustrate this concept, the following is a brief description of the antibody structure.

An antibody consists of two peptide chains, each containing a (light chain) or three (heavy chain) constant domain and a variable region (VL, VH) which in the different cases constitutes four FR regions and three spacers CDR. The antigen-binding site is formed by one or more CDRs, while the FR region provides the structural backbone of the CDRs and thus plays an important role in antigen binding. By altering one or more amino acid residues in the CDR or FR regions, it is customary for the skilled artisan to routinely produce mutant or diverse antibody sequences which, for example, can be screened for antigen with respect to novel or improved properties.

A more preferred embodiment of the invention is the antibody or antigen-binding fragment selected for the VH and VL sequences as shown in Table 9. Those skilled in the art can use the data in Table 9 to design peptide variants that fall within the scope of the present invention. Preferably, the variant is made by altering the amino acid in one or more of the CDR regions; the variant may also have one or more altered framework regions. Changes can also be in the skeleton area. For example, the peptide FR domain can be altered with a residue error compared to the germline sequence.

Alternatively, one skilled in the art can use the procedures described in, for example, Knappik A., et al., JMB 2000, 296: 57-86, by aligning the amino acid sequences disclosed herein with known sequences of antibodies of the same type. Compare the same analysis.

Furthermore, variants may be made by using one antibody as a starting point for optimization by making one or more amino acids in the antibody (preferably amino acid residues in one or more CDRs) Diversification, and by screening for variants with improved properties in combination with the resulting antibody variants. More preferably, one or more amino acid residues in the CDR3 of VL and/or VH are diversified. Diversification can be accomplished by synthesizing a set of DNA molecules using the Trinucleotide Mutation (TRIM) technique (Virnekäs B. et al., Nucl. Acids Res. 1994, 22: 5600.). An antibody or antigen-binding fragment thereof includes a molecule having a modification/variation including, but not limited to, a modification that causes a change in half-life (eg, modification of an Fc moiety or other attachment such as PEG molecule), modification of binding affinity change, or Modification of ADCC or CDC activity changes.

Examples of antibody variants provided are M048-D01 (TPP-1397, TPP-1398, TPP-1399, TPP-1400, TPP-1401, TPP-1402 and TPP-1403) as shown in Table 10, and M047- D08 (TPP-1406, TPP-1407, TPP-1408, TPP-1409, TPP-1410, TPP-1411, TPP-1412 and TPP-1415). The improved properties of these variant antibodies are shown in Table 11.

Conserved amino acid variant

Polypeptide variants can be made that retain the entire molecular structure of the antibody peptide sequences described herein. Providing the properties of individual amino acids, the skilled artisan will be able to identify some reasonable substitutions. Amino acid substitutions, i.e., "conservative substitutions" can be made, for example, based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphiphilicity of the residues involved.

For example, (a) a non-polar (hydrophobic) amino acid includes alanine, thioacid, isothioglycolic acid, valine acid, valine acid, phenylalanine, tryptophan and methionine; (b) Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, aspartic acid and glutamic acid; (c) positively charged (basic) amines The base acid includes arginine, lysine, and histidine; and (d) the negatively charged (acidic) amino acid includes aspartic acid and glutamic acid. Substitution is usually carried out between groups (a) - (d). Further, the acid and the valine can be substituted with each other based on their ability to destroy the α-helix. Similarly, certain amino acids such as alanine, cysteine, thioacid, methionine, aspartic acid, glutamic acid, histidine and lysine are more common in alpha-helices. And valine, isothioglycolic, phenylalanine, tyrosine, tryptophan and threonine are more common in β-sheets. Glycine, serine, aspartic acid, aspartic acid and proline are common at the turning point. Some preferred substitutions can be made in the following groups: (i) S and T; (ii) P and G; (iii) A, V, L and I. Providing known genetic codes, as well as recombinant and synthetic DNA techniques, can be easily constructed by those skilled in the art DNA of a conserved amino acid variant.

As used herein, "sequence identity" between two polypeptide sequences refers to the percentage of the same amino acid between the sequences. "Sequence homology" means the percentage of amino acids that are the same or represent a conservative amino acid substitution.

DNA molecule of the invention

The invention also relates to DNA molecules encoding the antibodies of the invention or antigen-binding fragments thereof. Such sequences include, but are not limited to, those shown in SEQ ID NOS: 3, 4, 13, 14, 23, 24, 33, 34, 43, 44, 53 and 54.

The DNA molecules of the invention are not limited to the sequences disclosed herein, but also include variants thereof. The DNA variants of the present invention can be illustrated by reference to their physical properties upon hybridization. Those skilled in the art will appreciate that using nucleic acid hybridization techniques, DNA can be used to identify complements and equivalents or homologs, and because DNA is a double strand. It should also be appreciated that hybridization may occur with less than 100% complementarity. However, to provide suitable selection of conditions, hybridization techniques can be used to distinguish DNA sequences based on their structural relevance to a particular probe. For guidance on these conditions, see Sambrook et al., 1989, supra, and Ausubel et al., 1995 (Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Sedman, JG, Smith, JA, & Struhl, K. eds. (1995). Current Protocols in Molecular Biology. New York: John Wiley and Sons).

The structural similarity between two polynucleotide sequences can be expressed as a function of the condition "stringency" under which the two sequences will hybridize to each other. As used herein, the term "stringent" means that the condition is not conducive to the degree of hybridization. Harsh

Conditions are strongly detrimental to hybridization, and only the most structurally related molecules will hybridize to each other under these conditions. Conversely, non-stringent conditions facilitate the expression of molecules with a lower degree of structural correlation to hybridize to each other. Therefore, hybridization severity is directly related to the structural correlation of the two nucleic acid sequences. The following relation for hybridization-based and associated with correlations (where a melting temperature T _m of a nucleic acid _{double-stranded):. A T m = 69.3} + 0.41 (G + C)%

b. T _m of double-stranded DNA with the number of mismatched base pairs every 1% decrease 1 ℃.

c.(T _m ) _μ2 -(T _m ) _μ1 =18.5 log ₁₀ μ2/μ1

Among them, μ1 and μ2 are the ionic strengths of the two solutions.

Hybrid stringency is a function of many factors, including total DNA concentration, ionic strength, temperature, probe size, and the presence of reagents that disrupt hydrogen bonding. Factors that enhance hybridization include high DNA concentration, high ionic strength, low temperature, longer probe size, and no reagents that disrupt hydrogen bonding. Hybridization usually takes place in two phases: the "combination" phase and the "washing" phase.

Functionally equivalent variant

Another class of DNA variants within the scope of the invention can be illustrated by reference to the products encoded thereby. These functionally equivalent polynucleotides are due to the degeneracy of the genetic code by which they are encoded in SEQ ID NO: 1, 2, 5-12, 15-22, 25-32, 35-42, 45-52, The same peptide sequence found in 55-60 was identified.

It is understood that the variants of the DNA molecules provided herein can be constructed in several different ways. For example, they can be constructed to fully synthesize DNA. Methods for efficiently synthesizing oligonucleotides ranging from 20 to about 150 nucleotides are widely available. See Ausubel et al ., section 2.11, Supplement 21 (1993). Overlapping oligonucleotides can be synthesized and assembled first in Khorana et al., J. Mol . Biol. 72: 209-217 (1971); also in the manner reported in Ausubel et al ., supra, section 8.2. Synthetic DNA is preferably designed using conventional restriction sites engineered at the 5' and 3' ends of the gene to aid in selection into a suitable vector.

As shown, the method of generating variants begins with one of the DNAs disclosed herein, followed by localization mutations. See Ausubel et al., supra, chapter 8, Supplement 37 (1997). In a typical method, the target DNA is selected into a single-stranded DNA phage vector. The single strand of DNA is isolated and hybridized to an oligonucleotide containing the desired nucleotide change (etc.). Synthetic complementary strands are synthesized and double-stranded phage are introduced into the host. Some of the resulting progeny will contain the desired mutant, which can be confirmed using DNA sequencing. In addition, various methods are available which increase the likelihood that the progeny phage will be the desired mutant. Such methods are well known to those skilled in the art and the kits used to generate such mutants are commercially available.

Recombinant DNA constructs and performance

The invention further provides recombinant DNA constructs comprising one or more nucleotide sequences of the invention. The recombinant construct of the present invention can be used together with a vector such as a plastid, phagemid, phage or viral vector into which a DNA molecule encoding an antibody of the present invention or an antigen-binding fragment thereof is inserted.

The antibodies, antigen binding portions or derivatives thereof provided herein can be prepared by recombinantly expressing a nucleic acid sequence encoding a light chain and a heavy chain or a portion thereof in a host cell. In order to express an antibody, an antigen-binding portion or a derivative thereof in a recombinant manner, DNA having a light chain and/or a heavy chain or a portion thereof may be used. The recombinant expression vector of the fragment is transfected into a host cell to express the light and heavy chains in the host cell. Nucleic acids encoding heavy and light chains are prepared and/or obtained using standard recombinant DNA methodology, such nucleic acids are incorporated into recombinant expression vectors and introduced into host cells, such as those described in Sambrook, Fritsch and Maniatis ( Eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989), Ausubel, FM et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and Boss et al. U.S. Patent No. 4,816,397.

Furthermore, a nucleic acid sequence encoding a variable region of a heavy chain and/or a light chain can be converted into, for example, a nucleic acid sequence encoding a full length antibody chain, Fab fragment or scFv. A DNA fragment encoding VL or VH is operably linked (so that the amino acid sequence encoded by the two DNA fragments is within the framework) to another DNA fragment encoding, for example, an antibody constant region or an elastic linker. Sequences of human heavy and light chain constant regions are known in the art (see, for example, Kabat, EA, el al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments including these regions can be obtained by standard PCR amplification.

In certain assays, the antibodies of the invention preferably be expressed as murine IgG, for example, immunohistochemistry using human samples can be more readily analyzed using murine antibodies. Thus, for example, the antibody Fab fragment M048-D01 (the VH chain of SEQ ID NO: 31, and the VL chain of SEQ ID NO: 32) is represented as murine IgG2a, referred to as M048-D01-mIgG2a (SEQ ID NO: 221) Heavy chain). This antibody was also used in Example 17 as a control.

To create a polynucleotide sequence encoding an scFv, a nucleic acid encoding VH and VL is operably linked to another fragment encoding an elastic linker such that the VH and VL sequences can be expressed as a continuous single chain protein, wherein the VL and VH regions are Linked by an elastic linker (see, for example, Bird et al. (1988) Science 242: 423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al., Nature (1990) 348: 552-554).

To express antibodies, antigen-binding portions or derivatives thereof, standard recombinant DNA expression methods can be used (see, for example, Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). For example, DNA encoding the desired polypeptide can be inserted into an expression vector, which in turn is transfected into a suitable host cell. Suitable host cells are prokaryotic and eukaryotic cells. Examples of prokaryotic host cells are, for example, bacteria, and examples of eukaryotic host cells are yeast, insect or mammalian cells. In some embodiments, the DNA encoding the heavy and light chains is inserted into separate vectors. In other embodiments, the DNA encoding the heavy and light chains is inserted into the same vector. It will be appreciated that the design of the expression vector (including the selection of regulatory sequences) is affected by factors such as the host cell chosen, the degree of expression of the desired protein, and the constitutive or inducible properties.

Bacterial performance

A useful expression vector for use in bacteria is by inserting a structural DNA sequence encoding the desired protein with an appropriate translation initiation and termination signal Operable reading orientation with a functional promoter. The vector will contain one or more phenotypic selectable markers as well as a replication source to ensure maintenance of the vector and, if desired, provide amplification in the host. Suitable prokaryotic hosts for transformation include different strains of Escherichia coli, Bacillus subtilis, Salmonella and Pseudomonas, Actinomycetes and Staphylococcus.

The bacterial vector can be, for example, based on phage, plastid or phagemid. Such vectors may contain a selectable marker and a bacterial origin of replication derived from a commercially available plastid (generally containing the known selection vector pBR322 (ATCC 37017)). After transformation of the appropriate host strain and growth of the host strain to the appropriate cell density, the selected promoter is inhibited/inducible by an appropriate means (e.g., temperature switching or chemical induction) and the cells are cultured for a further period of time. The cells are typically harvested by centrifugation, physically or chemically disrupted and the resulting crude extract is retained for further purification.

In bacterial systems, some expression vectors can be advantageously selected for the protein to be expressed depending on the intended use. For example, when a large amount of such proteins are to be produced to produce antibodies or to screen peptide libraries, a vector that directly expresses a high amount of fusion protein product that is readily purified will be desirable.

Thus, a specific example of the invention is an expression vector comprising a nucleic acid sequence encoding a novel antibody of the invention. See Example 2 for an illustrative description.

The antibodies of the invention or antigen-binding fragments thereof comprise naturally purified products, products of chemical synthesis procedures, and prokaryotic hosts (including, for example, Escherichia coli, Bacillus subtilis, Salmonella, and Pseudomonas, by means of recombinant techniques) Production of different strains of bacteria and staphylococci, preferably E. coli cells) Product.

Mammalian performance and purification

Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from large cell viruses (CMV) (such as CMV promoter/enhancement) Promoter and/or enhancer of simian virus 40 (SV40) (such as SV40 promoter/enhancer), adenovirus (such as adenovirus major late promoter (AdMLP)), and polyoma virus. For a more detailed description of the viral regulatory elements and their sequences, see, for example, U.S. Patent No. 5,168,062 to Stinski, U.S. Patent No. 4,510,245 to Bell et al., and U.S. Patent No. 4,968,615 to Schaffner et al. Recombinant expression vectors can also include a source of replication and a selectable marker (see, for example, U.S. 4,399,216, 4,634,665 and U.S. 5,179,017 to Axel et al.). Suitable selectable markers include assigning a gene that is resistant to a drug, such as G418, hygromycin or methotrexate, to a host cell into which the vector has been introduced. For example, the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate, while the neo gene confers resistance to G418. For efficient selection, the N-terminal 3 amino acids [EVQ] of the heavy chain (SEQ ID NO: 67 and SEQ ID NO: 69) may also be additionally expressed as [QVE], such as, for example, human IgG1. A variant of the heavy chain of M048-D01-hIgG1 (SEQ ID NO: 222). For efficient translocation, the N-terminus of the light chain can be extended by an amino acid residue such as alanine.

Transfection of expression vectors into host cells can be performed using standard techniques such as electroporation, calcium phosphate precipitation, and DEAE-polyglucose transfection.

For the expression of the antibodies, antigen-binding portions or derivatives thereof provided herein Suitable mammalian host cells include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, with DHFR selectable markers Used together (for example as described in RJ Kaufman and PA Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. In some embodiments, the expression vector is designed to be rendered The protein is secreted into the culture medium in which the host cells are grown. The antibody, antigen binding portion or derivative thereof can be recovered from the culture medium using standard protein purification methods.

The antibody of the present invention or antigen-binding fragment thereof can be recovered and purified from recombinant cell culture by known methods, including but not limited to ammonium sulfate or ethanol precipitation, acid extraction, protein A chromatography. Method, protein G chromatography, anion or cation exchange chromatography, phosphate-cellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and plant lectin Chromatography. It can also be purified by high performance liquid chromatography ("HPLC"). See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), for example, Chapters 1, 4, 6, 8, 9, and 10, each of which The manner of reference is incorporated herein.

Antibodies of the invention or antigen-binding fragments thereof include naturally purified products, products of chemical synthesis procedures, and products made from eukaryotic hosts (e.g., yeast, higher plants, insects, and mammals) by recombinant techniques. Depending on the host used in the recombinant production procedure, the antibodies of the invention may be glycosyl Or may be unglycosylated. These methods are described in many standard laboratory manuals, such as Sambrook, supra, sections 17.37-17.42; Ausubel, supra, chapters 10, 12, 13, 16, 18 and 20.

Thus, a particular embodiment of the invention is also a host cell comprising the vector or nucleic acid molecule, wherein the host cell can be a higher eukaryotic host cell (such as a mammalian cell), a lower eukaryotic host cell (such as a yeast cell) And may be a prokaryotic cell (such as a bacterial cell).

Another embodiment of the present invention is a method for producing an antibody and an antigen-binding fragment using the host cell, which comprises culturing the host cell under appropriate conditions and recovering the antibody.

Thus, another embodiment of the invention is the use of a host cell of the invention to produce an antibody (e.g., antibody M048-D01-hIgG1) according to the invention, and to purify such antibodies to at least 95% by weight homogeneity.

treatment method

The method of treatment involves administering to a subject in need of treatment a therapeutically effective amount of an antibody or antigen-binding fragment thereof contemplated by the present invention. A "therapeutically effective" amount is defined as the amount of antibody or antigen-binding fragment sufficient to deplete FGFR2-positive cells in a treated area of an individual - either in a single dose or in accordance with a multiple dose regimen, alone or in combination with other agents. It causes a reduction in adverse conditions, but the amount is toxicologically tolerable. The individual can be a human or non-human animal (eg, rabbit, rat, mouse, dog, monkey, or other lower primate).

The antibody of the invention or antigen-binding fragment thereof can be co-administered with a drug, and in some cases the antibody itself can be modified. For example, antibodies can be conjugated to cytotoxic agents or radioisotopes to further enhance Effectiveness.

The antibodies of the invention can be administered as a single agent or in combination with one or more other therapeutic agents, wherein the combination does not cause unacceptable adverse effects. The combination therapy comprises administering a single pharmaceutical dosage formulation comprising an antibody of the invention and one or more additional therapeutic agents, as well as the antibodies of the invention and each of the other therapeutic agents administered to their respective pharmaceutical dosage formulations. For example, the antibodies and therapeutic agents of the invention can be administered to a patient together in a single oral dosage composition, such as a lozenge or capsule, or each of the agents can be administered in separate dosage formulations.

When separate dosage formulations are used, the antibodies of the invention can be administered (e.g., simultaneously) with one or more other therapeutic agents at substantially the same time or administered at separate times (e.g., sequentially).

In particular, the antibodies of the invention may be immobilized or separately combined with other anti-tumor agents such as alkylating agents, antimetabolites, plant-derived anti-tumor agents, hormonal therapies, topologies Isomerase inhibitors, camptothecin derivatives, kinase inhibitors, targeting drugs, antibodies, interferons and/or biological response modifiers, anti-angiogenic compounds, and other anti-tumor drugs. In this regard, the following is a non-limiting list of second agent embodiments that can be used in combination with the antibodies of the invention: alkylating agents include, but are not limited to, nitrogen mustard N-oxide, cyclophosphamide (cyclophosphamide), ifosfamide, thiotepa, ranimustine, nimustine, temozolomide, altretamine, Apache Kun (apaziquone), clostatin (brostallicin), bendamustine, carmustine, estramustine, fotemustine, glufosfamide, equine Mafosfamide), bendamustin, and mitolactol; platinum-coordinated alkylating compounds include, but are not limited to, cisplatin, carboplatin, and iriplatin ( Eptaplatin), lobaplatin, nedaplatin, oxaliplatin, and satraplatin; anti-metabolites include, but are not limited to, methotrexate, 6- 6-mercaptopurine riboside, mercaptopurine, 5-fluorouracil alone or in combination with leucovorin, tegafur, deoxyfluorouridine (doxifluridine), carmofur, cytarabine, cytarabine ocfosfate, enocitabine, gemcitabine, fluoride Fludarabine, 5-azacitidine (5-azaciti) Dine), capecitabine, cladribine, clofarabine, decitabine, eflornithine, ethynylcytidine ), cytosine arabinoside, hydroxyurea, melphalan, nelarabine, nolatrexed, octadecyl phosphate, Disodium premetrexed, pentostatin, pelitrexol, raltitrexed (raltitrexed), triapine, trimetrexate, vidarabine, vincristine, and vinorelbine; hormonal therapies include, but are not limited to, Exemestane, Lupron, anastrozole, doxercalciferol, fadrozole, formestane, 11-beta steroid dehydrogenase 1 -beta hydroxysteroid dehydrogenase 1 inhibitors, 17-alpha hydroxylase/17,20 lyase inhibitors, such as abiraterone acetate, 5-alpha reductase inhibitors such as finasteride and epristeride, such as tamoxifen citrate and fulvestrant Antiestrogens, Trepstar, toremifene, raloxifene, lasofoxifene, letrozole, such as Bica Bicalutamide anti-androgen, flutamide, mifi Ketone (mifepristone), nilutamide, Casodex, and anti-lutein combined with it; plant-derived anti-tumor substances include, for example, those selected from mitotic inhibitors, such as Ebo Epothilones (such as sagopilone, ixabepilone and epothilone B), vinblastine, vinflunine, dosi Paclitaxel, and paclitaxel; Cytotoxic topoisomerase inhibitors include, but are not limited to, aclarubicin, doxorubicin, amonafide, belototecan, camptothecin , 10-hydroxycamptothecin, 9-aminocamptothecin, diflomotecan, irinotecan, topotecan, AI Edotecarin, epibbicin, etoposide, exatecan, gimatecan, lurototecan, bishydroxyindole ( Mitoxantrone), pirambicin, pixantrone, rubitecan, sobuzuxane, tafluposide, and combinations thereof; immunizing agents including interferon (such as interferon alpha, interferon alpha-2a, interferon alpha-2b, interferon beta, interferon gamma-1a and interferon gamma-nl), and other immunopotentiators such as L19-IL2 and other IL2 derivatives , filgrastim, lentinan, sizofilan, TheraCys, Ubumex (ube) Nimex), aldesleukin, alemtuzumab, BAM-002, dacarbazine, daclizumab, denileukin, gemtuzumab ), ozogamicin, ibritumomab, imiquimod, lenograstim, lentinan, melanoma vaccine (Corixa) ), molgramostim, sargramostim, tasonermin, tecleukin, thymalasin, Tositumomab, Vimlizin, epratuzumab, mitumomab, orgolovomab, pemtumomab, and Provenge; bioreactive modifiers are agents that modify the defense mechanisms or biological responses of biological organisms (such as the survival, growth or differentiation of tissue cells) such that they have antitumor activity; such agents include, for example, clouds Krestin, lentinan, sizofiran, picibanil, ProMune, and ubenimex; anti-angiogenic compounds including, but not limited to, Aqu Acitretin, aflibercept, angiostatin, aplidine, asentar, axitinib, bevacizumab , brivanib alaninat, cilengtide, combretastatin, endostatin, fenretinide, halofuginone , pazopanib, lanizumab, ranibizumab Rebimastat, recentin, regorafenib, removab, revlimid, sorafenib, squalamine, Sunitinib, telatinib, thalidomide, ukrain, vatalanib, and vitaxin; antibodies include, But not limited to trastuzumab, cetuximab, bevacizumab, rituximab Anti-rituximab, ticilimumab, ipilimumab, lumiliximab, catummaxomab, atacicicept, ovaviro Anti-oregovomab, and alemtuzumab; VEGF inhibitors such as, for example, sorafenib, regorafenib, bevacizumab, sunitinib (sunitinib), recentin, axitinib, aflibercept, telatinib, brivanib alaninate, vatalani (vatalanib), pazopanib, and ranibizumab; EGFR (HER1) inhibitors such as, for example, cetuximab, panitumumab, Viktor Ratio (vectibix), gefitinib, erlotinib, and Zactima; HER2 inhibitors such as, for example, lapatinib, trastuzumab ( Tratuzumab), and pertuzumab; mTOR inhibitors such as, for example, temsirolimus, sirolimus/rapamycin (Rapamy) Cin), and everolimus; c-Met inhibitor; PI3K and AKT inhibitor; CDK inhibitors, such as roscovitine and flavopiridol; spindle assembly checkpoint inhibition Agents and targeted anti-mitotic agents, such as PLK inhibitors, Aurora inhibitors (eg Hesperadin), checkpoint kinase inhibitors, and KSP inhibitors; HDAC inhibitors such as, for example, panobinostat, vorinostat, MS275, Bellin Nobel (belinostat), and LBH589; HSP90 and HSP70 inhibitors; proteasome inhibitors such as bortezomib and carfilzomib; serine/threonine kinase inhibitors, including MEK inhibition And Raf inhibitors, such as sorafenib; farnesyltransferase inhibitors such as, for example, tipifarnib; tyrosine kinase inhibitors, including, for example, dasatinib (dasatinib) ), nilotibib, regorafenib, bosutinib, sorafenib, bevacizumab, sunitinib , cediranib, axitinib, aflibercept, telatinib, imatinib mesylate, bofanini alainate (brivanib alaninate), pazopanib, ranibizumab, watt Talalanib, cetuximab, panitumumab, vectibix, gefitinib, erlotinib, rapa Latatinib, trastuzumab, pertuzumab, and c-Kit inhibitor; a vitamin D receptor agonist; a Bcl-2 protein inhibitor such as obobaclax, oblimersen sodium, and gossypol; a differentiation cluster 20 receptor antagonist such as For example, rituximab; a ribonucleotide reductase inhibitor such as, for example, gemcitabine; a ligand receptor 1 agonist that induces apoptosis in tumor necrotic cells, such as, for example, mapatumumab Serotonin receptor antagonists such as, for example, rEV598, xaliprode, palonosetron hydro-chloride, granisetron, Zindol, and AB-1001; integration Inhibitors include α5-β1 integrin inhibitors such as, for example, E7820, JSM 6425, volociximab, and endostatin; and male receptor antagonists include, for example, fluphenazine Nandrolone decanoate, fluoxymesterone, Android, Prost-aid, andromustine, bicalutamide, flutamide, apo-chlorine Apo-cyproterone, apo-flutamide (apo-flutamide), chlormadinone acetate, Androcur, Tabi, cyproterone acetate, and nilutamide; aromatase inhibitors such as, for example, anastrob Oxazole (anastrozole) Letrozole, testolactone, exemestane, aminoglutethimide, and formestane; matrix metalloproteinase inhibitors; other anti-cancer agents include, for example, Ali Retinoic acid (alitretinoin), ampericin (ampligen), atrasentan bexarotene, bortezomib, bosentan, calcitriol, sulfonic acid Exisulind, fotemustine, ibandronic acid, miltefosine, mitoxantrone, I-aspartate (I-) Asparaginase), procarbazine, dacarbazine, hydroxycarbamide, pegaspargase, pentostatin, tazaroten, valencene Velcade), gallium nitrate, canfosfamide, darinaparsin, and tretinoin.

In a preferred embodiment, the antibodies of the invention may be combined with chemotherapy (i.e., cytotoxic agents), anti-hormones, and/or target therapies (such as other kinase inhibitors (e.g., EGFR inhibitors), mTOR inhibitors, and angiogenesis inhibition. Agent) used in combination.

The compounds of the invention may also be used in combination with radiation therapy and/or surgical intervention in the treatment of cancer.

The antibodies of the invention or antigen-binding fragments thereof may in some cases be modified by themselves. For example, the antibody may be and is not limited to one or any of the above compounds Radioisotope combinations to further enhance utility. In addition, the antibodies of the invention may be used as such or in the compositions, in research and diagnostics, or as analytical reference standards and the like, which are well known in the art.

The antibodies of the invention or antigen-binding fragments thereof can be used as therapeutic or diagnostic tools in a variety of situations with abnormal FGFR2-signaling, such as cell proliferative disorders such as cancer or fibrotic diseases. Particularly suitable for the treatment of diseases and conditions by the antibodies of the invention are solid tumors, such as breast, respiratory, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid cancer and its distal end Transfer. These conditions also include lymphoma, sarcoma and leukemia.

Tumors of the digestive tract include, but are not limited to, the anus, colon, colorectal, esophagus, bladder, stomach, pancreas, rectum, small intestine, and salivary gland cancer.

Examples of esophageal cancer include, but are not limited to, esophageal cell carcinoma and adenocarcinoma, as well as squamous cell carcinoma, leiomyosarcoma, malignant melanoma, rhabdomyosarcoma, and lymphoma.

Examples of gastric cancer include, but are not limited to, intestinal type and diffuse gastric adenocarcinoma.

Examples of pancreatic cancer include, but are not limited to, ductal adenocarcinoma, adenoid squamous cell carcinoma, and pancreatic endocrine tumors.

Examples of breast cancer include, but are not limited to, triple-onset breast cancer, aggressive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ , and lobular carcinoma in situ (lobular carcinoma) Carcinoma in situ ).

Examples of respiratory cancer include, but are not limited to, small cell lung cancer and non-small Cell lung cancer, as well as bronchial adenoma and pleuropulmonary blastoma.

Examples of brain cancer include, but are not limited to, brain stem and hypothalamic neuroglioma, cerebellar and cerebral astrocytoma, glioblastoma, chorioblastoma, ependymoma, and neuroectoderm Pineal tumors.

Male reproductive organ tumors include, but are not limited to, prostate and testicular cancer. Tumors of the female reproductive organs include, but are not limited to, endometrial, cervical, ovarian, vaginal and vaginal cancer, and uterine sarcoma.

Examples of ovarian cancer include, but are not limited to, serous tumors, endometrial carcinomas, mucinous cystadenocarcinoma, granulosa cell tumors, selevar cell tumors, and male embryos.

Examples of uterine cancer include, but are not limited to, squamous cell carcinoma, adenocarcinoma, adenoid squamous cell carcinoma, small cell carcinoma, neuroendocrine tumor, vitreous cell carcinoma, and cervical villous adenocarcinoma.

Urinary tract tumors include, but are not limited to, bladder, penis, kidney, kidney, ureter, urethra, and hereditary and sporadic mastoid-like renal cell carcinoma.

Examples of renal cancer include, but are not limited to, renal cell carcinoma, urothelial cell carcinoma, proximal renal cell tumor (renal tumor), angiomyolipoma, renal eosinophilia, renal ureteral cancer, kidney Clear cell sarcoma, mesodermal nephroma and Wilm's tumor.

Examples of bladder cancer include, but are not limited to, transitional epithelial cell carcinoma, squamous cell carcinoma, adenocarcinoma, sarcoma, and small cell carcinoma.

Eye cancer includes, but is not limited to, intraocular melanoma and retinoblastoma.

Examples of liver cancer include, but are not limited to, hepatocellular carcinoma (hepatocellular carcinoma with or without a fibrolamellar variant), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular cholangiocarcinoma.

Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.

Head and neck cancer includes, but is not limited to, squamous cell carcinoma of the head and neck, pharynx, hypopharynx, nasopharynx, oropharyngeal cancer, lip and oral cancer, and squamous cell carcinoma.

Lymphomas include, but are not limited to, AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Birch's lymphoma, Hodgkin's disease, and lymphoma of the central nervous system.

Sarcomas include, but are not limited to, soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.

Leukemia includes, but is not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia. In a preferred embodiment, the antibody of the present invention or antigen-binding fragment thereof is suitable for use in the treatment and diagnosis of a cancer disease, which is included in the group consisting of gastric cancer, breast cancer, and pancreatic cancer. Colorectal cancer, kidney cancer, prostate cancer, ovarian cancer, cervical cancer, lung cancer, endometrial cancer, esophageal cancer, head and neck cancer, hepatocellular carcinoma, melanoma and bladder cancer. In addition, the antibodies of the invention or antigen-binding fragments thereof may also be used as a therapeutic or diagnostic tool in a variety of other conditions involving FGFR2, such as, but not limited to, fibrotic diseases (such as intrapulmonary fibrosis, sputum-induced lungs). Fibrosis, experimental lung fiber , primary fibrosis, renal fibrosis) and lymphatic smooth muscle hyperplasia, polycystic ovarian disease, acne, psoriasis, cholesteatoma, cholesteatoma chronic otitis media, periodontal disease, solar plaque, bowel Disease, atherosclerosis or endometriosis.

The above conditions have been well characterized in humans, but have similar causes in other animals, including mammals, and can be treated by administering the pharmaceutical compositions of the present invention.

For the treatment of any of the foregoing conditions, the pharmaceutical compositions for use in accordance with the present invention may be formulated using one or more physiologically acceptable carriers or excipients in a conventional manner. The antibodies of the invention or antigen-binding fragments thereof can be administered by any suitable means, which can vary depending on the type of disorder being treated. Possible routes of administration include parenteral (eg, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous), intrapulmonary and intranasal, and, if desired, for local immunosuppressive therapy, intralesional administration. Furthermore, the antibodies of the invention may be administered by pulse infusion, for example using a reduced dose of antibody. Preferably, the administration is by injection, preferably intravenous or subcutaneous injection, to some extent depending on whether the administration is short-lived or chronic. The amount to be administered will depend on various factors, such as clinical symptoms, individual weight, and whether or not to administer other drugs. Those skilled in the art will recognize that the route of administration will vary with the condition or condition being treated.

In accordance with the present invention, it is determined that the therapeutically effective amount of the novel polypeptide will depend, in particular, on the characteristics of the particular patient, the route of administration, and the nature of the condition being treated. For example, in the Open File of the International Coordinating Committee and Remington's Pharmaceutical Sciences, chapters 27 and 28, pp. 484-528 (18th ed., Alfonso Find general guidelines in R. Gennaro, Ed., Easton, Pa.: Mack Pub. Co., 1990). More specifically, determining the therapeutically effective amount will depend on factors such as drug toxicity and efficacy. Toxicity will be determined using methods well known in the art and as found in previous references. Efficacy will be determined using the same guidelines associated with the methods described in the examples below.

diagnosis method

The FGFR2 antibody or antigen-binding fragment thereof can be used to detect the presence of a tumor that exhibits FGFR2. The presence of FGFR2-containing cells or the shedding of FGFR2 in various biological samples (including serum and tissue biopsy samples) can be detected using FGFR2 antibodies. In addition, FGFR2 antibodies can be used in a variety of imaging methodologies, such as immunoscintigraphy using antibodies that bind to ⁹⁹ Tc (or other isotopes). For example, an imaging procedure similar to that described recently using ¹¹¹ In-conjugated anti-PSMA antibodies can be used to detect pancreatic or ovarian cancer (Sodee et al., Clin. Nuc. Med. 21:759-766, 1997). Another useful detection method is positron emission tomography by binding an antibody of the invention to an appropriate isotope (see Herzog et al., J. Nucl. Med. 34: 2222-2226, 1993).

Pharmaceutical composition and administration

A specific example of the invention is a pharmaceutical composition comprising an FGFR2 antibody or antigen-binding fragment thereof, alone or in combination with at least one other agent, such as a diazepam compound, which can be in any sterile, biocompatible pharmaceutical carrier (including, However, it is not limited to saline, buffered saline, dextrose and water. A further specific example is a FGFR2 antibody or antigen-binding fragment thereof and another pharmaceutical activation suitable for the treatment of FGFR2-related diseases such as cancer. Pharmaceutical composition of the compound. Either of these molecules can be administered alone or in combination with other agents, drugs or hormones into a pharmaceutical composition in which it is admixed with an excipient or a pharmaceutically acceptable carrier. In one embodiment of the invention, the pharmaceutically acceptable carrier is pharmaceutically inert.

The invention also relates to the administration of a pharmaceutical composition. These investments are achieved either orally or parenterally. Parenteral delivery methods include topical, intra-arterial (directly to tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal or intranasal administration. In addition to the active ingredient, such pharmaceutical compositions may contain a suitablepharmaceutically acceptable carrier which comprises an excipient and an adjuvant which facilitates processing the active compound into preparations for use in medicine. Further technical details regarding formulation and administration can be found in the latest version of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa.).

The pharmaceutical composition for oral administration can be used in a dosage formulation suitable for oral administration using a pharmaceutically acceptable carrier known in the art. These carriers enable the pharmaceutical compositions to be formulated into tablets, pills, dragees, sachets, liquids, gels, syrups, slurries, suspensions, and the like for ingestion by a patient.

The pharmaceutical composition for oral administration can be obtained by combining the active compound with a solid excipient, optionally grinding the resulting mixture and, after adding an appropriate auxiliary substance, if necessary, to obtain a lozenge or dragee core. . Suitable excipients are carbohydrate or protein fillers such as sugars (including lactose, sucrose, mannitol or sorbitol); starches of corn, wheat, rice, potato or other plants; celluloses such as methylcellulose, hydroxy Propyl methylcellulose or sodium carboxymethylcellulose; and gums, including Labo gum and tragacanth; and proteins such as gelatin and collagen. If necessary, a disintegrating or solubilizing agent such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate may be added.

The dragee core is provided with a suitable coating such as a concentrated sugar solution, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbomer gel, polyethylene glycol and/or titanium dioxide, fiber lacquer. A solution and a suitable organic solvent or solvent mixture. Dyestuffs or pigments may be added to the tablet or dragee coating for identification or identification of the amount of active compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol and sorbitol. The push-fit capsule may contain the active ingredient in admixture with a filler and a binder (such as lactose or starch), a lubricant (such as talc or magnesium stearate), and optionally a stabilizer. In soft capsules, the active compound can be dissolved or suspended in the presence or absence of a stabilizer, such as a fatty oil, liquid paraffin or liquid polyethylene glycol.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds. For injection, the pharmaceutical composition of the present invention can be formulated into an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and dextran. Furthermore, suspensions of the active compounds can be prepared in a suitable oily injection suspension. Suitable lipophilic solvents or vehicles include fatty acids such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. In addition, the suspension may also contain suitable stabilizers or increase the dissolution of the compound. A solution of the substance to allow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to the particular barrier to be penetrated can be used in the formulation. Such penetrants are generally known in the art.

Set

The invention further relates to a pharmaceutical package and kit comprising one or more containers filled with one or more of the aforementioned composition components of the invention. With respect to such containers (etc.), reference may be made to the form prescribed by the government agency for the manufacture, use or sale of a pharmaceutical or biological product, which means that the administrative agency approves the manufacture, use or sale of products for human investment.

In another embodiment, the kits can contain a DNA sequence encoding an antibody of the invention. Preferably, the DNA sequences encoding such antibodies are provided in a plastid suitable for transfection into a host cell and expressed by the host cell. The plastid may contain a promoter (usually an inducible promoter) to regulate the expression of the DNA in the host cell. The plastid may also contain appropriate restriction sites to facilitate insertion of other DNA sequences into the plastid to produce a variety of antibodies. These plastids may also contain many other elements that promote the selection and expression of the encoded protein. Such elements are well known to those skilled in the art and include, for example, selectable markers, initiation codons, stop codons, and the like.

Manufacturing and storage

The pharmaceutical compositions of the present invention can be made in a manner well known in the art, for example by conventional mixing, dissolving, granulating, dragee-making, milling, emulsifying, encapsulating, capturing or lyophilizing procedures.

The pharmaceutical composition can be provided as a salt and can be formed with an acid including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, Succinic acid, etc. Salts tend to be more soluble in aqueous or other protic solvents corresponding to the free base form. In other cases, a preferred preparation may be a lyophilized powder of 1 mM to 50 mM histidine, 0.1% to 2% sucrose, 2% to 7% mannitol in a pH range of 4.5 to 5.5, Combine with buffer before use.

After the pharmaceutical compositions comprising the compounds of the invention formulated in an acceptable carrier have been prepared, they can be placed in suitable containers and labeled for treatment of the indicated condition. With regard to administration of FGFR2 antibodies or antigen-binding fragments thereof, such labels can include amounts, frequencies, and methods of administration.

Effective dose

Pharmaceutical compositions suitable for use in the present invention include compositions comprising an active ingredient which is effective to achieve the desired purpose (i.e., the particular disease state characterized by the expression of FGFR2). Determining the effective dosage is well within the skill of the artisan.

With respect to any of the compounds, the therapeutically effective dose can be estimated initially in cell culture assays (e.g., tumor cells) or in animal models (usually mice, rabbits, dogs, pigs, or monkeys). Animal models are also used to achieve the desired concentration range and route of administration. This information can then be used to determine the dose and route of administration in humans.

A therapeutically effective dose means that the antibody or antigen-binding fragment thereof reduces the number of symptoms or conditions. Efficacy and toxicity of such treatment by standard pharmaceutical procedures in cell cultures or experimental animals of the assay compound, e.g. ED ₅₀ (50% of the population therapeutic effective dose) and LD ₅₀ (lethal to 50% of the dose groups) . The dose ratio between therapeutic utility and toxic utility is a therapeutic indicator, which can be expressed as the ratio ED ₅₀ /LD ₅₀ . Pharmaceutical compositions that exhibit high therapeutic indicators are preferred. The data obtained from cell culture analysis and animal studies are used to formulate dosage ranges for human consumption. Preferred dose of these compounds in not high ED ₅₀ or no toxicity within a range of circulating concentrations. The dosage varies within this range depending on the dosage employed, the severity of the patient, and the route of administration.

The exact dose is selected according to the individual patient to be treated according to the individual patient. The dosage and dosage are adjusted to provide a sufficient level of active moiety or to maintain the desired effect. Other factors that may be taken into account include the severity of the disease state, such as tumor size and location; age, weight and sex of the patient; diet, time and frequency of administration, drug combination, response sensitivity, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, weekly, or once every two weeks, depending on the half-life and clearance rate of the particular formulation.

Typical dosages can vary from 0.1 to 100,000 micrograms up to a total dose of about 2 g, depending on the route of administration. Guidance on specific dosages and delivery methods is provided in the references. See U.S. Patent No. 4,657,760; 5,206,344 or 5,225,212. Those skilled in the art will employ formulations that differ from the protein or its inhibitor in terms of polynucleotides. Similarly, delivery of a polynucleotide or polypeptide is specific to a particular cell, condition, location, and the like. The preferred specific activity range for radiolabeled antibodies is 0.1 to 10 mCi/mg protein (Riva et al., Clin. Cancer Res. 5: 3275-3280, 1999; Ulaner et al., 2008 Radiology 246(3): 895 -902).

The invention will be further illustrated by the following examples. For reference only The invention is provided by way of illustration of the invention. The exemplifications of the invention are not intended to limit the scope of the invention or the scope of the invention.

All of the examples are carried out using standard techniques known and conventional in the art, unless otherwise specified. The conventional molecular biotechnology of the following examples can be as described in standard laboratory manuals (such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). get on.

A preferred embodiment of the invention is:

A. An isolated antibody or antigen-binding fragment thereof, which is in a cell line SNU16 (ATCC-CRL-5974) and MFM223 (ECACC-98050130) which overexpress FGFR2 and a cell line AN3-CA (DSMZ) which exhibits mutant FGFR2 -ACC 267) and MFE-296 (ECACC-98031101) reduced cell surface expression of FGFR2 upon binding to FGFR2.

B. An isolated antibody or antigen-binding fragment thereof according to claim A, wherein the antibody or antigen-binding fragment thereof specifically binds to an extracellular N-terminal of FGFR2 as shown in (SEQ ID NO: 63) Bit ( ¹ RPSFSLVEDTTLEPE ¹⁵ ).

C. An isolated antibody or antigen-binding fragment thereof according to claim B, wherein the antibody binds to an extracellular N-terminal epitope (SEQ ID NO: 63) by at least one selected from the group consisting of The medium of the epitope residue of the group: Arg 1, Pro 2, Phe 4, Ser 5, Leu 6 and Glu 8.

D. The isolated antibody or antigen-binding fragment of any one of claims B to C, wherein the antibody or antigen-binding fragment thereof has at least one amine group of the N-terminal epitope of FGFR2 ( ¹ RPSFSLVEDTTLEPE ¹⁵ ) The acid residue becomes alanine and loses more than 50% of its ELISA signal a) the residue is selected from the group of Pro 2, Leu 6 and Glu 8 or b) the residue is selected from Arg 1 , Pro 2 , Phe 4 and the group of Ser 5 .

E. The antibody or antigen-binding fragment of any one of claims A to D, wherein the antibody or antigen-binding fragment competes for binding to FGFR2 with at least one antibody selected from the group consisting of: "M048-D01", M047-D08, M017-B02, M021-H02, M054-A05, M054-D03, TPP-1397, TPP-1398, TPP-1399, TPP-1400", "TPP-1401", "TPP-1402", "TPP-1403", "TPP-1406", "TPP-1407", "TPP-1408", "TPP-1409", "TPP- 1410", "TPP-1411", "TPP-1412" and "TPP-1415".

F. The antibody or antigen-binding fragment of claim E, wherein the amino acid sequence of the antibody or antigen-binding fragment is at least 50%, 55%, 60%, 70%, 80 with at least one of the following CDR sequences %, 90, or 95% identical: "M048-D01", "M047-D08", "M017-B02", "M021-H02", "M054-A05", "M054-D03", "TPP-1397" , "TPP-1398", "TPP-1399", "TPP-1400", "TPP-1401", "TPP-1402", "TPP-1403", "TPP- 1406", "TPP-1407", "TPP-1408", "TPP-1409", "TPP-1410", "TPP-1411", "TPP-1412" or "TPP-1415", or with the following VH Or the VL sequence is at least 50%, 60%, 70%, 80%, 90%, 92% or 95% identical: "M048-D01", "M047-D08", "M017-B02", "M021-H02", M054-A05, M054-D03, TPP-1397, TPP-1398, TPP-1399, TPP-1400, TPP-1401, TPP-1402, TPP -1403", "TPP-1406", "TPP-1407", "TPP-1408", "TPP-1409", "TPP-1410", "TPP-1411", "TPP-1412" or "TPP-1415" ".

G. The antibody or antigen-binding fragment of any one of claims E to F, wherein the antibody or antigen-binding fragment comprises at least one CDR sequence as shown in Table 9 and Table 10 or at least one variable weight Chain or light chain sequence.

H. An antibody or antigen-binding fragment according to claims A to G, comprising: a) a variable heavy CDR sequence as represented by SEQ ID NOs: 5-7 and represented by SEQ ID NOs: 8-10 a variable light chain CDR sequence, or b) a variable heavy CDR sequence as represented by SEQ ID NOs: 15-17, and a variable light chain CDR sequence represented by SEQ ID NOs: 18-20, or c) Variable heavy chain CDR sequences as represented by SEQ ID NOs: 25-27 and variable light chains represented by SEQ ID NOs: 28-30 a CDR sequence, or d) a variable heavy CDR sequence as represented by SEQ ID NOs: 35-37 and a variable light chain CDR sequence represented by SEQ ID NO: 38-40, or e) as SEQ ID NO: The variable heavy chain CDR sequences represented by 45-47 and the variable light chain CDR sequences represented by SEQ ID NOS: 48-50, or f) the variable heavy CDRs represented by SEQ ID NOS: 55-57 Sequences and variable light chain CDR sequences represented by SEQ ID NOS: 58-60, or g) variable heavy CDR sequences as set forth in SEQ ID NOS: 75-77 and by SEQ ID NOs: 78-80 a variable light chain CDR sequence represented, or h) a variable heavy CDR sequence as set forth in SEQ ID NOs: 85-87, and a variable light chain CDR sequence represented by SEQ ID NO: 88-90, or i a variable heavy CDR sequence as represented by SEQ ID NO: 95-97 and a variable light chain CDR sequence represented by SEQ ID NO: 98-100, or j) as represented by SEQ ID NOs: 105-107 Variable heavy chain CDR sequences and variable light chain CDR sequences represented by SEQ ID NOs: 108-110, or k) variable heavy chain CDR sequences as set forth in SEQ ID NOs: 115-117 and by SEQ ID NO: 118-120 is variable light a stranded CDR sequence, or 1) a variable heavy CDR sequence as represented by SEQ ID NOs: 125-127 and a variable light chain CDR sequence represented by SEQ ID NO: 128-130, or m) as SEQ ID NO The variable heavy chain CDR sequences represented by 135-137 and the variable light chain CDR sequences represented by SEQ ID NOs: 138-140, or n) the variable heavy chains represented by SEQ ID NOs: 145-147 CDR sequences and variable light chain CDR sequences represented by SEQ ID NOs: 148-150, or o) variable heavy CDR sequences as set forth in SEQ ID NOs: 155-157 and SEQ ID NO: 158-160 The variable light chain CDR sequences represented, or p) the variable heavy chain CDR sequences set forth in SEQ ID NOs: 165-167, and the variable light chain CDR sequences represented by SEQ ID NOs: 168-170, or q) a variable heavy CDR sequence as represented by SEQ ID NO: 175-177 and a variable light chain CDR sequence represented by SEQ ID NO: 178-180, or r) as set forth in SEQ ID NO: 185-187 The indicated variable heavy chain CDR sequences and the variable light chain CDR sequences represented by SEQ ID NOs: 188-190, or s) the variable heavy CDR sequences represented by SEQ ID NOs: 195-197 and by SEQ ID NO: variable light represented by 198-200 a stranded CDR sequence, or t) a variable heavy CDR sequence as represented by SEQ ID NO: 205-207 and a variable light CDR sequence represented by SEQ ID NO: 208-210, or u) as SEQ ID NO The variable heavy chain CDR sequences represented by 215-217 and the variable light chain CDR sequences represented by SEQ ID NOs: 218-220.

I. An antibody or antigen-binding fragment according to patent application AH, which comprises: a) a variable heavy chain sequence as represented by SEQ ID NO: 1 and a variable light chain sequence as represented by SEQ ID NO: 2, Or b) a variable heavy chain sequence as represented by SEQ ID NO: 11 and a variable light chain sequence as represented by SEQ ID NO: 12, or c) a variable heavy chain sequence as represented by SEQ ID NO: And a variable light chain sequence as represented by SEQ ID NO: 22, or d) a variable heavy chain sequence as represented by SEQ ID NO: 31 and a variable light chain sequence as represented by SEQ ID NO: 32, or e) a variable heavy chain sequence as represented by SEQ ID NO: 41 and a variable light chain sequence as represented by SEQ ID NO: 42, or f) a variable heavy chain sequence as represented by SEQ ID NO: 51 and a variable light chain sequence as represented by SEQ ID NO: 52, or g) a variable heavy chain sequence as represented by SEQ ID NO: 73 and a variable light chain sequence as represented by SEQ ID NO: 74, or h a variable heavy chain sequence as represented by SEQ ID NO: 83 and a variable light chain sequence represented by SEQ ID NO: 84, or i) a variable heavy chain sequence as represented by SEQ ID NO: 93 and a variable light chain sequence as represented by SEQ ID NO: 94, or j) a variable heavy chain sequence as represented by SEQ ID NO: 103 and a variable light chain sequence as represented by SEQ ID NO: 104, or k) a variable heavy chain sequence as represented by SEQ ID NO: 113 and ID NO: a variable light chain sequence represented by 114, or 1) a variable heavy chain sequence as represented by SEQ ID NO: 123 and a variable light chain sequence as represented by SEQ ID NO: 124, or m) The variable heavy chain sequence represented by SEQ ID NO: 133 and the variable light chain sequence represented by SEQ ID NO: 134, or n) the variable heavy chain sequence represented by SEQ ID NO: 143 and SEQ ID NO: a variable light chain sequence represented by 144, or o) a variable heavy chain sequence as represented by SEQ ID NO: 153 and a variable light chain sequence as represented by SEQ ID NO: 154, or p) SEQ. The variable heavy chain sequence represented by ID NO: 163 and the variable light chain sequence represented by SEQ ID NO: 164, or q) the variable heavy chain sequence represented by SEQ ID NO: 173 and SEQ ID NO: :1 a variable light chain sequence represented by 74, or r) a variable heavy chain sequence as represented by SEQ ID NO: 183 and a variable light chain sequence as set forth in SEQ ID NO: 184, or s) as SEQ ID NO a variable heavy chain sequence represented by 193 and a variable light chain sequence as represented by SEQ ID NO: 194, or t) a variable heavy chain sequence as represented by SEQ ID NO: 203 and The variable light chain sequence represented by SEQ ID NO: 204, or u) the variable heavy chain sequence represented by SEQ ID NO: 213 and the variable light chain sequence represented by SEQ ID NO: 214.

J. An antibody according to any one of the preceding claims, which is an IgG antibody.

K. An antigen-binding fragment according to any one of the preceding claims, which is a scFv, Fab, Fab' fragment or F(ab') ₂ fragment.

L. An antibody or antigen-binding fragment according to any one of the preceding claims, which is a monoclonal antibody or an antigen-binding fragment.

M. An antibody or antigen-binding fragment according to any one of the preceding claims, which is a human, humanized or chimeric antibody or antigen-binding fragment.

N. An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof as claimed in claims A to M.

O. An isolated nucleic acid sequence encoding an antibody or antigen-binding fragment as claimed in claims A to M.

P. A vector comprising the nucleic acid sequence of claim O.

Q. An isolated cell which exhibits an antibody or antigen-binding fragment according to any one of claims A to M and/or a nucleic acid as claimed in claim O or a carrier of patent application P.

R. The isolated cell of claim Q, wherein the cell is a prokaryotic cell or a eukaryotic cell.

S. A method of producing an antibody or antigen-binding fragment according to any one of claims A to M, which comprises culturing a cell as claimed in claim R and purifying the antibody or antigen-binding fragment.

T. An antibody or antigen-binding fragment as claimed in claims A to M or an antibody-drug conjugate as claimed in claim N, which is a medicament.

U. An antibody or antigen-antigen-binding fragment as claimed in claims A to M, which is used as a diagnostic agent.

V. An antibody or antigen-binding fragment as claimed in claims A to M or an antibody-drug conjugate as claimed in the scope of claim N as a medicament for the treatment of cancer.

W. A pharmaceutical composition comprising an antibody or antigen-binding fragment as claimed in claims A to M or an antibody-drug conjugate as claimed in claim N.

X. A pharmaceutical composition as claimed in the patent application and a combination of one or more therapeutically active compounds.

Y. A method of treating a condition or condition associated with an undesired presence of FGFR2, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition as claimed in the patent application or a combination of claim X.

Example Example 1: Production of antibodies from the n-CoDeR library Tools for phage selection:

Recombinant proteins used to isolate human antibodies of the invention were obtained from R&D Systems and are listed in Table 1. All variants used are unloaded The Fc-fusion protein is presented in the bulk preparation. hTRAIL-Fc acts as a depleting agent to avoid Fc linkers. Biotinylation of the protein was carried out using an excess of about 2 times molar biotin-LC-NHS (Pierce; Cat. No. 21347) according to the manufacturer's instructions and using a Zeba desalting column (Pierce; Cat. No. 89889) ) Go salt.

Regarding phage selection for cells, a human gastric cancer cell line KATO III (ATCC HTB-103) which exhibits naive FGFR2 on its cell surface was used.

Phage selection:

Isolation of a human antibody or antigen-binding fragment thereof of the present invention by using the naive Fab antibody library of BioInvent International AB n-CoDeR (Lund, Sweden; described in Söderling et al., Nat. Biotech. 2000, 18: 853-856) The phage display technology is a Fab library that derives all six CDRs. As summarized in Table 2, three different strategies were employed to select antibodies of the invention.

Table 2: Induction of selection strategies

The standard buffer used in this example was: 1x PBS: from Sigma (D5652-501)

PBST: 1x PBS supplemented with 0.05% Tween20 (Sigma, P7949)

Blocking buffer: PBST, supplemented with 3% BSA (Sigma A4503)

Precipitation buffer: 20% PEG (Calbiochem, 528877) in 2.5 M NaCl

FACS-buffer: PBS supplemented with 3% FBS (GIBCO, 10082) and 0.01% NaN ₃ (Sigma, 71289)

Briefly, aliquots of the Fab antibody library were centrifuged for 5 minutes at room temperature, and the resulting pellet was resuspended in 40 ml PBS and incubated for 1 hour on ice and centrifuged (4000 rpm) by adding precipitation buffer. Precipitated for the next hour). The pellet library was then resuspended in 1 ml blocking buffer and incubated for 30 minutes at room temperature.

At the same time, an aliquot of the amylin-coated Dynabead M280 (Invitrogen, 11206D) was prepared by washing on the end-to-end rotating device with PBS for 30 minutes three times. Thereafter, some aliquots were mixed with 200 nM biotinylated TRAIL-Fc protein while the remainder were mixed with the biotinylated protein as shown in Table 2. The mixture was incubated on an end-to-end rotating device for 30 minutes at room temperature followed by 3 washes in 1 ml PBS. The coated beads were finally blocked by resuspending in 1 ml blocking buffer and the beads were collected and the supernatant removed.

To deplete the undesired Fc linker, the blocked library (described above) was added to the TRAIL-Fc coated blocked Dynabead and incubated for 30 minutes at room temperature with rotation. After the beads were collected on a magnetic stand, the supernatant was mixed with blocked Dynabead coated with the target protein. After incubation for 60 minutes on the end-to-end rotating device, the samples were washed 3 times with blocking buffer followed by 5 times with PBST. Bound phages were eluted by the addition of 100 μl of triethanolamine solution (TEA, 100 mM). After incubation for 10 minutes at room temperature, the sample was neutralized by the addition of 400 μl of 1 M Tris-Cl, pH 7.5.

The panning strategy I included 2 rounds of panning of whole cells as the source of the target protein (see Table 2). To this end, KATO III cells were resuspended in ice-cold FACS buffer at a density of 10 ⁷ cells/ml. An aliquot of the retrieved phage was added to 1 ml of cell suspension and incubated at 4 °C by end-to-end rotation. Next, the cells were washed 10 times with 2.5 ml of FACS buffer, followed by elution of the bound phage with 300 μl of 76 nM citric acid (pH 2.5). After 5 minutes of incubation, the cells were centrifuged at 400 g and 4 ° C for 5 minutes, and the supernatant was neutralized by adding 300 ml of 1 M Tris-Cl, pH 7.5.

Eluted phage were propagated and phage titers were determined as previously described (Cicortas Gunnarsson et al., Protein Eng Des Sel 2004; 17(3): 213-21. Briefly, aliquots of the elution solution were retained. In the potency experiment, while the rest were used to transform the exponentially growing E. coli TG1 (from Stratagene) for the preparation of new rounds, third and fourth rounds of selection according to the strategy shown in Table 2. Phage stocks. For each selection round, the input and output phage systems were titrated against exponentially growing E. coli TG1 and selected from rounds 2 to 4 for analysis in phage ELISA.

Enzyme linked immunosorbent assay (ELISA): Phage ELISA:

Selected phage selected from different rounds were analyzed for specific use of phage ELISA. Briefly, by adding 10 μl of overnight culture (in LB-medium supplemented with 100 μg/ml ampicillin (Sigmal, A5354), 1% glucose) to 100 μl of fresh medium (supplemented with 100 μg/ml Anbi) Xilin and 0.1% glucose (Sigma, G8769) in LB-medium) and incubated in 96-well MTP at 250 rpm and 37 °C for phage display until OD600 reached 0.5. Then 40 μl of the helper phage M13KO7 (Invitrogen, 420311) was added and the samples were incubated for 15 minutes at 37 ° C without shaking. After addition of IPTG (f.c. of 0.5 mM; final volume of 200 μl), the cells were incubated overnight at 30 ° C while shaking at 200 rpm.

A 96-well ELISA plate pre-coated with avidin (Pierce, 15500) was coated with 1 μg/ml of biotinylated FGFR2-2β Fc (IIIb) or biotinylated TRAIL-Fc overnight at 4 °C. The plate was washed 3 times with PBST the next day, blocked with reagent treatment, and washed again 3 times with PBST. At the same time, the phage culture was simply centrifuged, then 125 μl of the supernatant was removed and 125 μl of blocking buffer was mixed. 100 μl of blocked phage was then transferred to each well and incubated for 1 hour at room temperature. After washing 3 times with PBST, an anti-M13 antibody (GE Healthcare, 27-9421-01; 1:2500 diluted in PBST) coupled to HRP was added and incubated at room temperature for 1 hour. The color reaction was colored by the addition of 50 μl of TMB (Invitrogen, 2023) and was stopped after 5 to 15 minutes by the addition of 50 μl of H ₂ SO ₄ (Merck, 1120801000). The colorimetric reaction was recorded in a disc reader (Tecan) at 450 nM.

Screening for sFab by ELISA:

For the production of soluble Fab fragments (sFab), the phagemid DNA was selected by the third and fourth rounds and digested with restriction enzymes EagI (Fermentas, FD0334) and EcoRI (NER, R0101L) according to the supplier's instructions to remove the gene. III sequence. The resulting fragments were recombined and the construct was transformed into a chemical competent E. coli Top 10 using standard methods. Individual isolates were picked and transferred to 96-well plates containing LB-medium (100 μg/ml ampicillin (Sigma, A5354), 1% glucose) and shaken at 250 rpm and 37 °C. The next morning, 10 ml of the preculture was transferred to 150 μl of fresh LB-medium (100 μg/ml ampicillin (Sigma, A5354), 0.1% glucose) until the OD600 reached 0.5. Thereafter, sFab production was induced by the addition of IPTG (f.c. 0.5 mM) and incubation was continued overnight at 30 ° C while shaking at 200 rpm. The next morning, 50 μl of BEL-buffer (24.7 g/l boric acid, 18.7 g/l NaCl, 1.49 g/l EDTA pH 8.0, 2.5 mg/ml lysozyme (Roche)) was added to each well at room temperature. The mixture was incubated for 1 hour. Next, add the tool 1/3 volume of blocking buffer with 9% BSA and an additional 30 minute incubation step at room temperature, except for detection using an anti-couple to HRP (1:2500 dilution; Sigma; A 0293) -hIgG (Fab-specific) 50 μl of each well was analyzed for sFab binding to the target in an ELISA essentially as described for phage.

Example 2: Small-scale production of soluble Fab screening results

The unique screening results were produced on a small scale for the initial analysis of their binding to different FGFR-protein variants (see Example 3). 20-50 ml of LB-medium (supplemented with 0.1 mg/ml ampicillin and 0.1% glucose) was inoculated into a preculture of individual E. coli Top 10 strains containing unique Fab sequences that were cloned into the original pBIF-vector. However, the gene III sequence is absent. The sFab was induced to produce by the addition of 0.5 mM IPTG (final concentration) and incubated overnight at 30 °C at 250 rpm for overnight incubation.

Thereafter, the cells were harvested by centrifugation and lysed by lysis buffer (containing 20% sucrose (w/v), 30 mM TRIS, 1 mM EDTA, pH 8.0, 1 mg/ml lysozyme at 4 ° C (Sigma L- Incubation was carried out for 1 hour in 6876) and 2.5 U/ml benzonase (Sigma E1014), followed by careful addition of an equal volume of PBS. Thereafter, the cleared supernatant was applied to Dynabead for His-marker separation (Invitrogen, 101-03D) and incubated on an end-to-end rotating device for 2 hours at 4 °C. Next, the substrate was washed 3 times with buffer 1 (50 mM Na-phosphate buffer, pH 7.4, 300 mM NaCl, 5 mM imidazole, 0.01% Tween-20), then in buffer 2 (containing 0.005% Tween-20). a washing step in PBS). Finally, in buffer E (10 mM Na-phosphate buffer, pH 7.4, 300 mM NaCl, Fab was eluted with 300 mM imidazole) and concentrated in Vivaspin 50 (cutoff value 10000; from GE; 28-9322-25) using PBS-buffer. The protein content of the Fab was analyzed and the purity was analyzed by SDS-PAGE.

Example 3: Cross-reactive pattern of antibodies

Unique screening results were produced on a small scale as described in Example 2 and tested for binding to the different FGFR-variants listed in Table 3 in ELISA.

All variants used presented as Fc-fusion proteins in unsupported preparations. The protein was biotinylated using an excess of about 2 times the molar biotin-LC-NHS (Pierce; Cat. No. 21347) according to the manufacturer's instructions. Salt was removed using a Zeba demineralized column (Pierce; Cat. No. 89889).

For ELISA, a 96-well plate pre-coated with avidin (Pierce, 15500) was coated overnight with 1 μg/ml biotinylated protein at 4 °C. A well coated with biotinylated TRAIL-Fc was used as a reference. The plate was washed 3 times with PBST the next day, blocked with reagents, and washed 3 times with PBST. 100 μl of purified Fab (1 μg/ml) was added and incubated for 1 hour at room temperature. After washing 3 times with PBST, anti-hIgG (Fab-specific) coupled to HRP (1:2500 dilution: Sigma; A 0293) was added and incubated for 1 hour at room temperature. The color reaction was colored by the addition of 50 μl of TMB (Invitrogen, 2023) and was stopped after 5 to 15 minutes by the addition of 50 μl of H ₂ SO ₄ (Merck, 1120801000). The colorimetric reaction was recorded in a disc reader (Tecan) at 450 nM. Wells containing TRAIL-Fc were used as background values and the signals relative to the background were calculated as summarized in Table 4.

Signal ratio to background: 0:<2;+:2-3;++:3-5;+++:>5

As shown in Table 4, the antibodies of the present invention bind to human and murine FGFR2, regardless of the alpha and beta and IIIb and IIIc spliced forms. As shown in Table 4, the antibodies of the present invention did not bind to FGFR1, FGFR3 and FGFR4.

Example 4: FGFR2 antibody binds to the cell surface of cancer cell lines

To determine the binding characteristics of anti-FGFR2 antibodies to mouse, rat and human cancer cell lines, a panel of cell lines was tested for binding by flow cytometry. The adherent cells were washed twice with PBS (without Ca and Mg) and separated by cell-free dissociation buffer (Invitrogen) based on enzyme-free PBS. Cells at approximately 10 ⁵ cells / well were suspended in FACS buffer (without Ca / Mg of PBS, Biochrom, containing 3% FCS, Biochrom) in. The cells were centrifuged (250 g, 5 min, 4 °C) and the supernatant was discarded. The cells were resuspended in a dilution of the antibody of interest in FACS buffer (5 μg/ml in 80 μl unless otherwise indicated) and incubated on ice for 1 hour. Next, the cells were washed once with 100 μl of cold FACS buffer and 80 μl of secondary antibody (PE goat anti-human IgG, Dianova #109-115-098, or PE goat anti-mouse IgG) diluted 1:150 was added. , Jackson Immuno Research #115-115-164). After incubation for 1 hour on ice, the cells were again washed with cold FACS buffer, resuspended in 100 μl of FACS buffer and analyzed by flow cytometry using FACS-Array (BD Biosciences). Results were calculated as the geometric mean detected by the antibody of interest minus background fluorescence detected using a separate secondary antibody. The values were scored according to the following system: geometric mean - geometric mean of individual secondary antibodies >10:+, >100:++, >1000:+++, 10000:++++, close to () Category boundaries in . List of cross-reactivity profiles of antibody strains used for antibodies:

As shown in Table 5, all anti-FGFR2 antibodies of the invention used at a concentration of 5 μg/ml bind to a wide range of murine (4T1, EMT6), rat (RUCA) and humans exhibiting FGFR2 (in the table) Tumor cells derived from all other cell strains included.

(geometric mean - geometric mean of individual secondary antibodies >10:+, >100:++, >1000:+++, >10000:++++, close to the category boundary in ())

To determine antibody binding to EC ₅₀ values of selected cancer cell lines, as described above to FGFR2 antibody stained cells, but with different concentrations of antibody, based range 0.1-100 nM. EC ₅₀ values were determined based Graph Pad Prism software and use are presented in Table 6. The three antibodies with the highest affinity (M017-B02-hIgG1, M048-D01-hIgG1, M047-D08-hIgG1) were shown in human (SNU-16, MFM223), murine (4T1) and rat (Ruca) cell lines. Below the EC ₅₀ value of the nanomolar to low naim. M021-H02-hIgG1, M054- A05-hIgG1 and M054-D03-hIgG1 also show low values of ₅₀ nM EC cells in murine and human cell lines. Thus, all test antibodies are cross-reactive when bound to human, murine and rat cells that express FGFR2.

(nd means not measured / measured)

Example 5: Epitope mapping by Pepscan Chemical Linked Peptide (CLIPS) technology for scaffolding

To determine the binding properties of the antibodies found, a thorough epitope map was performed based on the peptide-based peptide scanning (Pepscan) proprietary chemical-linked peptide (CLIPS) technique (Timmerman et al., J. Mol. Recognit. 2007, 20). :283-99). A total of 8653 CLIPS peptides of 15AA and 30AA length were designed for naive human FGFR2, including linear, conformational and discontinuous epitopes. The peptide was synthesized on a peptide array. The antibodies of the invention were tested on a peptide array in a human IgGl format analyzed by ELISA-based. Peptides with the highest ELISA values were analyzed to identify consensus amino acid sequences.

To reconstruct the discrete epitopes of the target molecule, a peptide library was constructed. This was done using Pepscan proprietary chemical link peptide (CLIPS) technology (Timmerman et al., J. Mol. Recognit. 2007, 20: 283-99). The CLIPS technology allows peptides to be constructed into monocyclic, bicyclic, tricyclic, sheet-like folds, helical folds, and combinations thereof. The CLIPS template is coupled to a cysteine residue. The polycysteine side chain in the peptide is coupled to one or two CLIPS templates. For example, a solution of 0.5 mM T2 CLIPS template 1,3-bis(bromomethyl)benzene is dissolved in ammonium bicarbonate (20 mM, pH 7.9) / acetonitrile (1:1 (v/v)). This solution was added to the peptide array. The CLIPS template was ligated into two cysteine side chains as presented in a solid phase binding peptide such as a peptide array (455 well plates, 3 μl wells). Carefully shake the peptide array in the solution for 30 to 60 minutes while completely covering the solution. Finally, the peptide array was extensively washed with an excess of H ₂ O and sonicated for 30 minutes at 70 ° C in a disruption buffer (β-mercaptoethanol in 1% SDS/0.1% PBS (pH 7.2)). The sonication is then repeated for 45 minutes in H ₂ O. Peptides with T3 CLIPS but now with three cysteines were made in a similar manner.

Antibody binding to individual peptides was tested in a PEPSCAN-based ELISA (Slootstra et al., Molecular Diversity 1996, 1: 87-96). The peptide array was pre-incubated with 5% to 100%-binding buffer (1 hour, 20 °C). The binding buffer consisted of 1% Tween-80, 4% horse-serum, 5% ovalbumin (w/v) and was diluted with PBS. After washing, the peptide array was incubated with a primary antibody solution (1 to 5 μg/ml) in PBS (containing 1% Tween-80) (overnight at 4 °C). After washing, the peptide array was incubated for 1 hour (anti-human) in a 100% binding buffer of antibody peroxidase conjugate at 1/1000 dilution at 25 °C. After washing, the peroxidase was added with 2,2'-methazo-di-3-ethylbenzothiazoline sulfonate (ABTS) and 2 μl/ml of 3% H ₂ O ₂ . After 1 hour, the color was measured. Color formation was quantified using a charge coupled device (CCD)-camera and image processing system.

data processing

The raw data is the optical value obtained by the CCD-camera. Values range from 0 to 3000 mAU, similar to a standard 96-well plate ELISA-reader. Combine the values for analysis. Occasionally a hole contains air bubbles, causing a pseudo-positive value, manually checking the card and scoring any value due to air bubbles to zero.

All antibodies of the invention bind to the same epitope, which contains the N-terminal residue of FGFR2 ( ¹ RPSFSLVEDTTLEPE ¹⁵ ). Analysis of 1257 CLIPS with linear peptides showed the same high ELISA values for the N-terminal peptide.

The N-terminal residue ( ¹ RPSFSLVEDTTLEPE ¹⁵ ) is present in all splice variants of human FGFR2 and is independent of D3 alternative splicing to generate the IIIb and IIIc isoforms (see Figure 1). This epitope is also present if domain D1 is cleaved from full-length FGFR2 ((SEQ ID NO: 61; FGFR2 a) to produce a shorter beta form of FGFR2 (SEQ ID NO: 62). In this case, the epitope Just before the domain D2 (see Figure 1).

Of particular interest, the N-terminal sequence is conserved in humans, mice, rats, and rhesus monkeys. This can create cross-reactivity between species.

This novel epitope is outside the known ligand binding site and the heparin binding site (see Figure 1) and produces novel properties of the antibodies of the invention.

Example 6: Precise mapping of epitopes based on peptide alanine scanning

To define the binding properties of the antibodies of the invention in more detail, an alanine-scan was performed. 15AA and 30AA length peptides were synthesized as described in Example 5 and the individual amino acids of the human FGFR2 sequence were replaced with alanine residues for one peptide. The binding of the antibodies was analyzed as described in Example 5. If the replacement of the amino acid residue with alanine results in a significant decrease in the binding signal, then the residue is believed to be critical for binding.

Table 7 shows the key residues for the N-terminal portion of FGFR2 ( ¹ RPSFSLVEDTTLEPE ¹⁵ ) against the antibodies of the invention.

(Remarks for residues that are critical for binding (X). Lose more than 50% of the ELISA signal by changing this residue to alanine)

The antibodies M048-D01 and M021-H02 are of particular interest because they bind to the position Ser-5 regardless of the variation. This enables them to additionally bind to human, mouse, rat and rhesus FGFR2 (SEQ ID NO: 63), rabbit (SEQ ID NO: 64), pig (SEQ ID NO: 65) and dog (SEQ ID NO) :66) FGFR2, and more species can be used for preclinical development.

Example 7: Antibody affinity of N-terminal epitopes analyzed by Biacore

To limit the binding affinity of the N-terminal peptide to the epitope, a Biacore surface plasma resonance experiment was performed.

The binding affinity of the anti-FGFR2 antibody was determined by surface plasma resonance analysis on a Biacore T100 instrument (GE Healthcare Biacore, Inc.). An antibody such as human IgG1 was immobilized on a CM5 detection wafer via an indirect capture reagent (anti-human IgG (Fc)). Reagents using the "Human Antibody Capture Kit" (BR-1008-39, GE Healthcare Biacore, Inc.) as described by the manufacturer. Approximately 5000 RU monoclonal mouse anti-human IgG (Fc) antibody was immobilized per well. Anti-FGFR2 antibody was injected at a concentration of 5 μg/ml at 10 μl/min for 10 seconds. Peptides of various concentrations (400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.12 nM) in HEPES-EP buffer (GE Healthcare Biacore, Inc.) derived from different species (human The first 15 FGFR2 of mouse, rat, rhesus FGFR2 (SEQ ID NO: 63), rabbit (SEQ ID NO: 64), pig (SEQ ID NO: 65) and dog (SEQ ID NO: 66) The amino acid was injected through the immobilized anti-FGFR2 antibody for 3 minutes at a flow rate of 60 μl/min and allowed to dissociate for 5 minutes. Reference cell correction was performed on the line followed by buffer sample deduction to obtain a sensory map. The dissociation equilibrium constant (K _D ) is calculated by the ratio of k _on ) to the dissociation rate (k _off ) constant, which is obtained by fitting the sensing map to the first-order 1:1 binding model using the Biavaluation software (version 4.0). .

M048-D01-hIgG1 and M047-D08-hIgG1 to K _D value of about 100 nM binding human, murine, rat and rhesus the FGFR2 (Please refer to Table 8). As supported by alanine scanning, M048-D01 for all peptides derived from several species shows the same K _D values (see Table 8).

Example 8: Stimulation of P-FGFR2 (phosphorylated FGFR2) in a cell line overexpressing FGFR2 after short-term incubation with anti-FGFR2 antibody

To determine the effect of anti-FGFR2 antibodies on phosphorylated FGFR2 (P-FGFR2) cell concentration after short-term incubation, a P-FGFR2 ELISA was performed. MFM223 cells were plated in 960 cells per well in 96-well plates in growth medium (MEM Earle (Biochrom; F0315) + 10% FCS + 2 mM glutamic acid). 24 hours after the plate was placed, the cells were incubated with the antibody (10 μ/ml) for 15 minutes, followed by two washing steps using PBS and dissolved in 100 μl of cold lysis buffer (containing 50 mM Hepes pH) by shaking for 5 minutes. 7.2, 150 mM NaCl, 1 mM MgCl ₂ , 10 mM Na ₄ P ₂ O ₇ , 100 mM NaF, 10% glycerol, 1.5% Triton X-100 with freshly added complete protease inhibitor cocktail (Roche No. 1873580001), 4 mM Na ₃ VO ₄ , pH was adjusted to 7.4) using NaOH. The samples were snap frozen and stored at -80 °C until analysis. Measurement of P-FGFR2 concentration was performed using R&D Systems' P-FGFR2 ELISA kit according to the manufacturer's instructions. OD (Tecan Spectra, Rainbow) was measured at 450 nM and background correction was performed. The concentration of P-FGFR2 was calculated as the % untreated control concentration. Parallel samples were incubated with the same isotype of non-cell binding control IgG as compared to the non-specific utility of the antibody format.

The results are shown in Figure 2 and indicate that the anti-FGFR2 antibodies M048-D01-hIgG1 and M047-D08-hIgG1 significantly induced P-FGFR2 concentrations. In contrast, control IgG antibodies or anti-FGFR2 antibodies (MAB665, MAB684, MAB6843) commercially available from R&D did not show any significant effect on P-FGFR2 concentrations after short-term incubation. These results reveal the efficacious effect of the anti-FGFR2 antibody described in the present invention on FGFR2 after short-term incubation.

Example 9: Cells overexpressing FGFR2 after long-term incubation with anti-FGFR2 antibody desensitize P-FGFR2 due to stimulation of FGF7

To determine the utility of anti-FGFR2 antibodies for cell concentration of phosphorylated FGFR2 (P-FGFR2) after long-term incubation and the utility of antibody treatment for FGF7-induced FGFR2 phosphorylation, a P-FGFR2 ELISA was performed. MFM223 cells were plated in 96-well plates in growth medium (MEM Earle (Biochrom; F0315) + 10% FCS + 2 mM glutamic acid) at 7000 cells per well. 24 hours after the plate was laid, the cells were incubated with the antibody (10 μ/ml) for 24 minutes, followed by incubation for 15 minutes in the presence or absence of FGFR7 (R&D Systems, 25 ng/ml). The cells were then washed twice with PBS and dissolved in lysis buffer (50 mM Hepes pH 7.2, 150 mM NaCl, 1 mM MgCl ₂ , 10 mM Na ₄ P ₂ O ₇ , 100 mM NaF, 10% glycerol, by shaking for 5 minutes at room temperature). 1.5% Triton X-100 was mixed with freshly added complete protease inhibitor cocktail (Roche No. 1873580001), 4 mM Na ₃ VO ₄ , using NaOH to adjust the pH to 7.4). Samples were snap frozen and stored at -80 °C until analyzed by R&D's P-FGFR2 ELISA according to the manufacturer's instructions. Optical density (Tecan Spectra, Rainbow) was measured at 450 nM and background correction was performed. The concentration of P-FGFR2 was calculated as the % untreated control concentration. Parallel samples were incubated with the same isotype of non-cell binding control IgG as compared to the non-specific utility of the antibody format.

The corresponding results are presented in Figure 3. FGF7 stimulation of P-FGFR2 concentration was increased approximately 4-fold in cells that were not treated with cells and in cells treated with isotype control IgG. In contrast, in samples pretreated with anti-FGFR2 antibodies, FGF7 induced only a concentration of P-FGFR2 of 1.37 to 1.4 fold.

In summary, these results show that culturing cells with the anti-FGFR2 antibody of the present invention desensitizes the stimulation of FGF7.

Example 10: Cell line down-regulates FGFR2 surface expression after incubation with anti-FGFR2 antibody

To analyze the surface expression of FGFR2 after treatment with anti-FGFR2 antibody, FACS analysis was performed in different cell lines with overexpression of FGFR2 (MFM223, SNU16) or FGFR2 mutations (AN3-CA, MFE-296). Adherent cells were washed twice with PBS (no Ca and Mg) and separated by cell-free dissociation buffer (Invitrogen) based on enzyme-free PBS. The cells were suspended in 80 μl of growth medium at approximately 0.5*10 ⁵ cells/well (MFM226, MFE-296: MEM Earle (Biochrom; F0315) + 10% FCS + 2 mM glutamic acid, SNU-16: RPMI 1640 (Biochrom , FG1215) + 10% FBS, AN3-CA: MEM Earle (Biochrom; FG0325) + 10% FCS + 1 mM sodium pyruvate + 1 x NEA: non-essential amino acid Biochrom K0293). 20 μl of a 5-fold concentrated antibody dilution (final concentration of 10 μg/ml) was added and incubated at 37 ° C for 4.5 hours. At the end of the incubation period, cells were washed once with 100 μl of FACS buffer and stained with antibody (at 5 μg/ml for mouse anti-FGFR2 for hIgG and human anti-FGFR2 for mIgG) at 4 °C. Minutes were followed by additional washing with 100 μl of FACS buffer. PE-stained secondary antibody (PE goat anti-human IgG, Dianova #109-115-098, or PE goat anti-mouse IgG, Jackson Immuno Research #115-115-164, via 1) was added in a volume of 80 μl : 150 dilution), incubated at 4 ° C for 45 minutes and analyzed by flow cytometry using a FACS array (BD Biosciences) after additional washing with FACS buffer. In a control experiment, the anti-system that competes for overlapping epitopes is excluded by incubation with the antibody of interest and the corresponding detection antibody. The geometric mean measured after staining with the secondary antibody alone was subtracted from the geometric mean of the peaks measured after staining with anti-FGFR2 antibody. Results were calculated as % control cells incubated for 4.5 hours in the absence of antibody.

The results are shown in Figure 4. Incubation of cells with control IgG showed no decrease in the surface performance of FGFR2, while anti-FGFR2 antibodies M048-D01-hIgG1 and M047-D08-hIgG1 down-regulated the surface concentration of FGFR2 in all 4 cell lines to 39-60% and overexpressed with FGFR2. Performance or mutation has nothing to do. In contrast, there are no other anti-FGFR2 antibodies or antibodies commercially available from R&D (MAB665, MAB684, MAB6843) or those described elsewhere (GAL-FR21, AL-FR22; WO2010/054265 and Zhao et al. Clin Cancer Res. 2010, 16: 5750-5758)) showed down-regulation of surface FGFR2 in all four cell lines regardless of FGFR2 overexpression or mutation. GAL-FR21 downregulates the surface concentration of FGFR2 in cells expanded with FGFR2 (SNU16 and MFM223), but has no effect on cell lines carrying the FGFR2 mutation. GAL-FR22 achieved down-regulation of surface expression of 73 and 21% FGFR2 in FGFR2 mutant cell lines (AN3-CA and MFE-296, respectively), but had no significant effect on surface FGFR2 concentration in SNU16 and MFM223 cells. MAB684 and MAB6843 in turn caused a decrease in the surface concentration of about 60% of FGFR2 in the FGFR2 mutant cell line and had no significant effect on the cell line overexpressing FGFR2. Finally, MAB665 did not show any effect at all on the FGFR2 surface concentration.

In summary, the anti-FGFR2 antibodies M048-D01-hIgG1 and M047-D08-hIgG1 are the only anti-FGFR2 antibodies that induce downregulation of FGFR2 surface in cancer cell lines, regardless of FGFR2 overexpression or mutation.

Example 11: Downregulation of total FGFR2 concentration in cancer cells after long-term incubation with anti-FGFR2 antibody

To analyze whether down-regulation of FGFR2 surface induced by anti-FGFR2 antibody would result in a long-term decrease in total FGFR2 concentration, the total protein concentration of FGFR2 was analyzed by FGFR2 ELISA. SNU16 cells were plated at 5,000 cells/well in 96-well plates in growth medium (RPMI 1640 (Biochrome, FG1215) + 10% FBS). After 2 hours, cells were incubated with different concentrations of the indicated anti-FGFR2 antibodies or corresponding isotype control IgG. 96 hours after the start of antibody incubation, the cells were centrifuged at 300 g for 5 minutes at room temperature, washed twice with PBS and shaken for 5 minutes at room temperature and 100 μl of lysis buffer (50 mM Hepes pH 7.2, 150 mM NaCl) was added. , 1 mM MgCl ₂ , 10 mM Na ₄ P ₂ O ₇ , 100 mM NaF, 10% glycerol, 1.5% Triton X-100 with freshly added complete protease inhibitor cocktail (Roche No. 1873580001), 4 mM Na ₃ VO ₄ , used The NaOH was adjusted to pH 7.4 to dissolve. Samples were snap frozen and stored at -80 °C until analysis using the total-FGFR2-ELISA kit (R&D Systems) according to the manufacturer's instructions. Optical density (Tecan Spectra, Rainbow) was measured at 450 nM and background correction was performed simultaneously. To calculate the absolute concentration of total FGFR2, a standard curve using the isolated FGFR2 protein was applied according to the manufacturer's (R&D Systems) recommendations. Results are expressed as % FGFR2 concentration measured in control cells incubated for 96 hours in the absence of antibody.

The results are presented in Figure 5. Incubation with the anti-FGFR2 antibody of the present invention for 96 hours resulted in a reduction in total FGFR2 concentration of 41 to 55%. A half maximal reduction was achieved at a dose of 3 μg/ml anti-FGFR2 antibody. In contrast, incubation with isotype control antibodies had no effect on total FGFR2 concentrations.

Taken together, these results indicate that the anti-FGFR2 antibodies M048-D01-hIgG1 and M047-D08-hIgG1 not only cause a short-term decrease in surface FGFR2 concentration, but also a long-term decrease in total FGFR2 concentration.

Example 12: Internalization of anti-FGFR2 antibodies into cells

These antibodies are analyzed for their ability to internalize after binding to the FGFR2 antigen by the anti-FGFR2 antibody of the invention.

To see this process, FGFR2-specific antibodies M048-D01-hIgG1 and M047-D08-hIgG1 were selected as well as isotype control antibodies. The antibodies were bound at pH 8.3 in the presence of a two molar excess of CypHer 5E mono-NHS ester (batch 357392, GE Healthcare). After binding, the reaction mixture was dialyzed (slide-A-Lyser Dialysis Cassettes MWCD 10 kD, Fa. Pierce) overnight at 4 °C to eliminate excess dye and adjust the pH. The protein solution was then concentrated (VIVASPIN 500, Fa Sartorius stedim biotec). In addition to the pH dependent fluorescent dye CypHer 5E, the ph-independent dye Alexa 488 was used. The dye loading of the antibody was determined using a spectrophotometer (Fa. NanoDrop). The dye loading of M048-D01-hIgG1 and M047-D08-hIgG1 with the isotype control (M014) was in a similar range. The affinity of the labeled antibody is tested in a cell binding assay to ensure that the label does not alter binding to FGFR2. These labeled antibodies were used in the following internalization assays. Prior to treatment, cells (2 x 10 ⁴ /well) were seeded into 100 μl of medium in 96-MTP (fat, black clear bottom, No 4308776, Fa. Applied Biosystems). After incubation for 18 hours at 37 ° C / 5% CO 2 , the medium was changed and labeled anti-FGFR2 antibodies M048-D01-hIgG1 and M047-D08 were added at different concentrations (10, 5, 2.5, 1, 0.3, 0.1 μg/ml). -hIgG1. The same treatment was carried out with an isotope control antibody (negative control). The incubation time was selected to be 0, 5 hours, 1 hour, 2 hours, 3 hours, 6 hours, and 24 hours. Fluorescence measurements were performed using an InCellAnalyzer 100 (Fa. GE Healthcare). Particle count and total fluorescence intensity were determined in a kinetic manner.

High specificity and significant internalization of M048-D01-hIgG1 and M047-D08-hIgG1 were observed in cancer cell lines SNU16 (stomach cancer) and SUM52PE (breast cancer) expressing endogenous FGFR2.

This internalization was dependent on the type of restriction because uptake was only possible with anti-FGFR2 antibodies, while no internalization was observed using isotype controls. Anti-FGFR2 antibodies showed a 20 to 40-fold increase in antibody internalization compared to the isotype control during the first 6 hours. The isotype control showed a little internalization after prolonged exposure (>24 hours).

Internalization of the Alexa 488-labeled anti-FGFR2 antibody after binding revealed that more than 50% of the internalized antibodies appeared to follow the endocytic pathway.

In Fig. 6, M048-D01-hIgG1 and M047-D08-hIgG1 showed microscopic evaluation of the timing of specific endocytosis after binding to cells expressing endogenous FGFR2. The antibody (2.5 μg/ml) of the breast cancer cell line SUM 52PE was internalized. The particle count was determined kinetically. on Fast internalization was observed for M048-D01-hIgG1 and M047-D08-hIgG1, while the isotype control hIgG1 was not internalized.

A more detailed assessment of the delivery path was performed using co-staining of small G-proteins. Rab GTPase regulates several steps of conjunctival transport (including vesicle formation, vesicle movement along the actin and tubulin network, and membrane fusion). To distinguish the different pathways, two Rab proteins were selected to stain -Rab7, which is expressed in late phagosomes and lysosomes, and Rab11, which is expressed early in the regenerative phagosomes. After internalization of the labeled antibody for 6 hours, the cells were fixed and permeabilized with methanol before staining with Rab 7 and Rab11 antibodies. The results are shown in Figure 7.

M048-D01-hIgG1 and M047-D08-hIgG1 showed significant co-staining with Rab 7, whereas co-staining with Rab 11 was only a few. These results indicate that after internalization of FGFR2, the complex enters the phagosome-lysosomal pathway.

The staining patterns of other such antibodies (such as GAL-FR21 and GAL-FR22 (WO2010/054265 and Zhao et al. (Clin Cancer Res. 2010, 16: 5750-5758)) appear to be completely different. Little staining was detected here using Rab7, but more co-staining was achieved using Rab11. This indicates that these antibodies internalize upon binding to the FGFR2 receptor and prefer a recovery pathway.

Example 13: Test of anti-FGFR2 antibody of the present invention in experimental tumor of mouse model

The in vivo potency of the anti-FGFR2 antibodies of the invention is tested, for example, by a subcutaneous allogeneic or homologous tumor model. Experts know the previous skill method to prove The efficacy of the antibodies of the invention. For example, mice are thus subcutaneously inoculated with tumor cells expressing the underlying FGFR2. Thereafter, the tumor bearing mice were treated with an antibody of the invention targeting FGFR2, a non-binding isotype control or phosphate-buffered saline (PBS). The antibody was administered intraperitoneally or intravenously twice a week. To test for additive anti-tumor efficacy, the FGFR2 Ab of the invention is combined with common standard care and compared to single agent efficacy. Tumor growth is monitored by frequent measurement of tumor area by calipers. After tumor growth and treatment for several weeks, tumors were harvested and the tumor weight or tumor size (calculated by the length x width) of animals treated with the anti-FGFR2 antibody of the present invention was compared with those treated with PBS or isotype control antibody. . Mice treated with the anti-FGFR2 antibody of the present invention exhibited significantly smaller tumors.

Human or murine tumor cells expressing FGFR2 are subcutaneously implanted into the flanks of immunocompromised mice (eg, nude or SCID mice). Each mouse was detached from the cell culture flask by 0.25-10 million cells, centrifuged and suspended in 100 μl PBS, 50% medium/50% matrigel or 100% matrigel, respectively. The cells were then subcutaneously inoculated under the skin of the mouse flank. In a patient-derived tumor model, tumors collected from gastric cancer patients were subcutaneously passed through immunocompromised mice. For the efficacy of testing anti-FGFR2 antibodies, tumor blocks of defined size (2 x 2 mm) were transplanted subcutaneously into the flanking flank of mice. A tumor was identified within a few days. If the tumor reaches the size of 20 mm ² (cell-derived tumor) or 100 mm ² (patient-derived tumor), the treatment begins as early as the tumor area (mm ² ) is calculated by the length x width and by the length x Tumor volume (mm ³ ) was calculated at a width of ² /2. Treatment with antibodies is carried out intraperitoneally or intravenously via tail vein injection. The anti-system was dissolved in PBS or 50 mM sodium acetate, 150 mM NaCl. The antibody was administered in a volume of 10 ml/kg. The treatment schedule is based on the pharmacokinetic behavior of the antibody. As a standard, antibodies were administered twice a week (alternating every third and fourth day). As a standard, treatment was performed until the control group reached the maximum possible tumor size. Or stop processing earlier. As a standard, 8 mice were used per treatment group. The number of mice per treatment group can be increased if a higher variation in tumor growth is expected. Parallel to the treatment group, the control group was treated with PBS according to the same treatment schedule. During the study, tumor area was frequently evaluated by measuring the length and width of the tumor using a caliper. At the end of the study, tumors were collected and weighed. The ratio of the average tumor weight of the antibody-treated group (T) to the average tumor weight (C) of the control was expressed as T/C. If the treatment group and the control group were stopped at different time points or the tumor weight could not be used as a read value and became necrotic, the T/C ratio was calculated based on the tumor area at the last common measurement time point.

Two Mio human gastric cancer SNU-16 cells in 50% medium/50% Matrigel were inoculated subcutaneously into the flanks of female nodSCID mice. When the tumor reached an average size of 20-30 mm ² , treatment with anti-FGFR2 antibody was initiated intraperitoneally and continued twice a week until the end of the study. If the tumor in the control group reached the maximum acceptable size, the study was discontinued and tumors were weighed and weighed.

All of the tested anti-FGFR2 antibodies of the invention significantly reduced tumor growth compared to controls. T/C was produced by treatment with M017-B02-hIgG1, M021-H02-hIgG1, M048-D01-hIgG1, M054-A05-hIgG1, M054-D03-hIgG1, and M047-D08-hIgG1 at a dose of 2 mg/kg. They are 0.19, 0.22, 0.17, 0.19, 0.21 and 0.22 (see Figures 8 to 13).

The subjects 2.5x10 ⁵ 4T1 murine breast cancer cells were inoculated subcutaneously into NMRI nu / nu mice side flank in the 100% PBS. Isogenic mice were selected to replace immunocompromised mice to avoid the development of neutralizing antibodies against human IgG proteins. Tumors were treated at the time point when the tumor had reached an average size of 24 mm ² . To test the possible additive anti-tumor effect of M048-D01-hIgG1, treatment with M048-D01-hIgG1, lapatinib or paclitaxel alone, and with M048-D01-hIgG1 in combination with paclitaxel or lapatinib Mouse. As a standard, mice were treated with PBS alone. Intravenous (iv) treatment was performed twice a week using M048-D01-hIgG1, lapatinib was administered orally once a week (po) and paclitaxel was intravenously once a week. All processing is done until the end of the study. Since the tumor became necrotic at the end of the study, the tumor area was used on day 13 after tumor cell inoculation to determine anti-tumor efficacy. This study revealed that the combination of M048-D01-hIgG1 with lapatinib or paclitaxel achieved additive anti-tumor efficacy: monotherapy with lapatinib versus paclitaxel did not significantly alter tumor growth compared to vehicle control, respectively. However, M048-D01-hIgG1 alone caused a significant decrease compared to the carrier, and its T/C was 0.73. The combination with lapatinib and paclitaxel reduced this T/C to 0.58 and 0.52, which was statistically significant relative to monotherapy (see Figures 14 and 15).

Sources derived from patients, 2×2 mm gastric tumor masses from GC10-0608 and GC12-0811 (Prof. Huynh Hung, National University of Singapore (NUS)) were passed through to immunocompromised mice, subcutaneously transplanted to females with insufficient immune function Naive mice. Tumor size was frequently assessed using a caliper to measure the tumor in two dimensions and the tumor volume was calculated by the length x width ² /2. Different doses of M048-D01-hIgG1 treatment were started at the time point when the tumor reached an average size of about 100 mm ³ . Intravenous (iv) treatment was performed twice daily at doses of 5, 2 and 1 mg/kg M048-D01-hIgG1. In the tumor model with high FGFR2 protein expression (GC10-0608), based on the final tumor weights of 0.55, 0.60 and 0.41, all three doses caused a significant decrease in T/C tumor growth (see Figure 16). In a model with significantly lower FGFR2 protein (GC12-0811), 5 and 2 mg/kg of M048-D01-hIgG1 caused a significant reduction in tumor weight, resulting in a T/C of 0.70 and 0.67 (see Figure 17). . Consistent with the lower FGFR2 performance, 1 mg/kg of M048-D01-hIgG1 did not result in a significant reduction in final tumor weight. Regarding the treatment of two other tumor models, the breast cancer model MFM223 and the colorectal cancer model NCI-H716 (ATCC-CCL-251), we did not find an appropriate administration protocol that significantly reduced tumor growth.

Example 14: Down-regulation of P-FGFR and total FGFR2 concentrations in xenograft tumors after treatment with anti-FGFR2 antibody

The down-regulation observed for the analysis of total FGFR2 concentration and the simultaneous decrease in P-FGFR2 were also seen in xenograft tumors in vivo, and SNU-16 tumors were analyzed by Western blot after treatment with anti-FGFR2 antibody. Tumors were collected at the end of the xenograft experiments in which NOD/SCID mice were treated twice weekly with anti-FGFR2 antibody 2 mg/kg ip (see Example 13 for details). Tumors were obtained 24 hours after the last injection of antibody, snap frozen in liquid nitrogen and stored at -80 °C until analysis. Frozen Western tumors were cut into approximately 5 mm diameter slices and placed in pre-cooled 5 mm steel beads (Qiagen) and 500 μl lysis buffer (50 mM Hepes pH 7.2, 150 mM NaCl, 1 mM) prior to Western blot analysis. MgCl ₂ , 10 mM Na ₄ P ₂ O ₇ , 100 mM NaF, 10% glycerol, 1.5% Triton X-100 with freshly added complete protease inhibitor cocktail (Roche No. 1873580001), 4 mM Na ₃ VO ₄ , using NaOH The pH was adjusted to 7.4) in a 2 ml centrifuge tube. The samples were lysed in a Tissuelyzer (Qiagen) at 300 Hz for 3 minutes followed by incubation on ice for 30 minutes. Next, the samples were centrifuged at 13,000 rpm for 10 minutes at 4 ° C in a Micro-centrifuge (Eppendorf), and the supernatants of the sections from the original tumor were pooled together. Tumor lysis protein concentration was determined in the product by using a BCA Protein Assay Kit (Novagen, lysates were diluted 1:50 in H ₂ O in). The sample was diluted to a final concentration of 5 mg/ml and 50 μl of the sample was mixed with 7.7 μl of (10*) sample reducing agent and 19.2 μl (4*) NuPAGE sample buffer (Invitrogen). A sample corresponding to 115 μg of protein was applied to a NuPAGE 4-12% SDS page gel of Invitrogen and run at 120 V for 2 hours and 45 minutes. Dyeing was performed by the iBlot system (Invitrogen) according to the manufacturer's recommendations. Membranes were blocked for 2 hours at room temperature in PBST in 5% BLOT QuickBlocker (Invitrogen), followed by overnight incubation with primary antibody at 4 °C. The primary antibody system was as follows: P-FGFR: #AF3285, R&D Systems, 0.5 μg/m; total FGFR2: M017-B02-hIgG1, 4 μg/ml in 3% BLOT QuickBlocker in PBST. The next day, the membrane was washed three times in PBST, followed by secondary antibody (peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L) at room temperature (Jackson ImmunoResearch #111-035-003 or peroxidase- The cells were incubated for 2 hours in combination with AffiniPure goat anti-human IgG + IgM (H+L) (Jackson ImmunoResearch #109-035-127, 1:10000 in 3% BLOT QuickBlocker/PBST). Then, the membrane was washed with PBST for 10 minutes 4 Secondly, the signal was detected by chemiluminescence after incubation with the ECL reagent. To detect the loading control, the membrane was disassembled by shaking the solution (1:10 in Milipore-H2O) for 15 minutes at room temperature. , then blocked and detected using 1:1000 anti-actin antibody #A2066 (Sigma) in 3% QuickBlocker/PBST.

Representative results for each group of 2 animals treated with anti-FGFR2 antibodies are shown in Figure 18, alongside the control IgG treated animal samples. After treatment with the anti-FGFR2 antibody of the present invention, the total FGFR2 and P-FGFR concentrations were strongly reduced. Therefore, the mode of action of the down-regulation of total FGFR2 in an in vitro study is also related to allograft tumors treated with the anti-FGFR2 antibody of the present invention.

Example 15: Subcutaneous xenograft cancer model using antibody drug conjugates

Anti-FGFR2 antibodies can be conjugated to cytotoxic small molecules using procedures well known in the art (e.g., Liu et al., Proc Natl. Acad. Sci. (1996), 93, 8618-8623). A431 cells were maintained as adherent cultures in DMEM supplemented with 10% FBS. NOD SCID or other immunocompromised 6 to 7 week old mice will be subcutaneously inoculated with 1-5 x 10e6 cells in 0.1 ml of medium on the right side. When the tumor size reached about 25 mm ² , the antibody drug conjugate was administered intraperitoneally 3 times every 4, 7 or 10 days at a dose of 1 to 10 mg/kg. Control mice will be treated with PBS or an irrelevant monoclonal antibody (with the same toxic group) and the tumor size measured twice weekly with a sliding caliper. Anti-tumor efficacy was assessed by treating tumor size relative to control treatment against anti-FGFR2 antibody drug conjugates.

Example 16: Production of mature variants of selected antibodies with increased affinity

The anti-FGFR2 antibodies of the invention, which were discovered by phage display as shown in Table 9, will be further optimized by affinity maturation.

Antibody affinity maturation is a two-step process in which a combination of saturation mutations and stable high-throughput screening to identify small amounts of mutations that cause increased affinity. In the first round of affinity maturation, according to BMC Biotechnology 7:65, the location diversity of wild-type antibodies was based on the use of NHK-trinucleotide 定位 by positional mutation (where N represents 25% mixed adenine, thymine, bird) Each of 嘌呤 and cytosine nucleotides, and K represents 50% of each of the mixed thymine and guanine nucleotides, was introduced. Thus, all 20 amino acids are introduced at the individual amino acid sites. This position randomization is limited to six complementarity determining regions (CDRs). In the second round of affinity maturation, beneficial substitutions were recombined and screened for further enhancement. Examples of each variant are shown in Table 10.

Two different types of ELISA were used to determine the binding enhancement of mutant variants: a) Peptide binding ELISA: a synthetic peptide containing a C-terminus linked to biotinylated lysine RPSFSLVEDTTLE PEG-Ttds-Lys (biotin) ( The peptide sequence is derived from the amino acid sequence of the epitope of SEQ ID NO: 63, synthesized by JPT Peptide Technology GmbH, Berlin, Germany, and b) the recombinant protein binding ELISA: recombinant human FGFR2 (DNA sequence of human FGFR2 ( NP_000132.3) Met 1-Glu 377, fused to a polyhistidine acid tag at the C-terminus, #10824-H08H, Sino Biological Inc., Beijing, China).

Briefly, 20 μl of anti-human IgG Fc specificity (# I2136; sigma) in coating buffer (# 121125 Candor Bioscience GmbH) was coated at 2.2 μg/ml in two ELISA formats at 37 °C. The MTP plate (384 well Maxisorp, Nunc) was allowed to last for 2.5 hours. After a washing step using 50 μl PBST (phosphate buffered saline, 137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ , pH 7.4, 0.05% Tween 20), at 20 to The plate was blocked with 50 μl of 10% Smart Block (#113500, Candor Bioscience GmbH) for 1 hour at 22 ° C and the washing step was repeated 3 times. Incubation of 20 μl for 1 hour at 20 to 22 ° C in 0.035 μg/ml (peptide-based assay) or 0.2 μg/ml in 10% Smart Block of PBST (based on recombinant human FGFR2 protein) The concentration of the anti-FGFR2 variant was fixed, depending on the form and variant to be analyzed. After one washing step with 50 μl PBST, 20 μl of a quadruplet serially diluted in 10% Smart Block of PBST was added at a maximum concentration of 100 nM and incubated for 1 hour at 20 to 22 ° C, and Repeat the washing step 3 times. To detect biotinylated epitope peptides, 20 μl of avidin/POD conjugate (#S5512, Sigma) diluted 1:1000 in PBST in 10% SmartBlock was administered at 20 to 22 °C for 1 hour. To detect recombinant FGFR2 protein, 20 μl of anti-His/HRP conjugate (#71840, novagen) diluted 1:10000 in 10% SmartBlock in PBST was administered at 20 to 22 °C for 1 hour. After 3 washing steps, add 20 μl of 10 μM amplex red substrate (# A12222, Invitrogen) in 50 mM sodium hydrogen phosphate, pH 7.6 and detect the fire using a common fluorescence reader (eg Tecan M1000) Optical signal. EC50 values were assessed by fitting the data (S-type dose-response, variable slope, bottom set to background; GraphPad Prism software).

Several examples of variants with amino acid substitutions produced in the heavy and light chains of M048-D01 (TPP-1403) are provided in Table 10. All variants were evaluated in two ELISA formats with different antigenic forms compared to the non-CDR variants, showing a strong increase in antigen binding (Table 11).

Several examples of variants with amino acid substitutions produced in the heavy and light chains of M047-D08 (TPP-1415) are provided in Table 10. All variants showed significant improvement in antigen binding compared to variants with non-CDR changes (Table 11).

Differences between the two forms regarding numerical results, EC50, and enhancement factors are unexpected, but may be interpreted as the use of antigens in the form of peptides or proteins and the formation of a final detectable enzyme conjugate on top of the ELISA sandwich. Difference: The K _{D of the} anti-His-HRP conjugate and the His-tagged FGFR2 are unknown, but it is likely to be several times higher than the K _D (10-15 M) of biotin and avidin used to detect the epitope peptide. Magnitude. Therefore, the sensitivity of the binding peptide is significantly higher than that of the His-marker protein, enabling the measurement of a smaller EC50. Additionally or alternatively, the difference may be due to an error in the interaction of the anti-FGFR2 antibody with the two antigens, although they are identical in a stretch of 15 amino acids; first, the chemistry of the C-terminal posterior portion of the molecule is very different The next 15 amino acids may adopt a different 3D configuration than the corresponding region in the FGFR2 protein. Both interpretations may be related to differences between peptide- and protein-based ELISAs, showing smaller EC50 values in peptide ELISA formats.

The data in Table 11 clearly indicates that M048-D01 (TPP-1403) binds to FGFR2 at its N-terminal sequence, as indicated in the epitope peptide, and several variants with amino acid substitutions in the CDRs are unexpected. The ground is also, even with a higher affinity. It is noted that the substitution N102I is present in five of the six other variants of TPP-1403, along with the number in CDR-L1, CDR-L2, CDR-L3, CDR-H2 and/or CDR-H3 A further substitution, but not in TPP-1399, unexpectedly showed a per-amine acid (K) at position HC-102.

The data in Table 11 indicates that M047-D08 (TPP-1415) binds to FGFR2 at its N-terminal sequence, as indicated in the epitope peptide, and several variants with amino acid substitutions in the CDRs unexpectedly Also, even with a higher affinity. The M047-D08 (TPP-1415) variant with multiple amino acid substitutions showed a binding increase of about 4 to 40 fold, with TPP-1409 (2.1 nM) being the least and TPP-1406 (0.22 nM) being the most. It should be noted that three of them have one G102L (TPP-1406, -1407, and -1412) and one G102V (TPP-1408) substitution, accompanied by CDR-L1, CDR-L2, CDR-L3, Several other substitutions in CDR-H1 and/or CDR-H3.

In addition, the internalization potency of the mature anti-FGFR2 antibody was summarized in Table 11. The enhancement factor is calculated based on the total particle strength/cell produced by the internalization and decomposition of the mature antibody relative to the corresponding value of the parent antibody. The same finding was obtained by comparing the particle counts/cells in the same antibody. Experimental details are described in Example 12. It is to be noted that all mature variants of M048-D01 (TPP-1403) showed an increase in internalization efficiency (1.9 to 2.4 fold). The M047-D08 (TPP-1415) variant TPP-1412 showed a 1.5-fold increase in internalization potency. Internalization is an important property of the antibodies of the invention.

In the variants provided for M047-D08 and M048-D01, it is clear that variants of these antibodies may have similar or enhanced properties if the epitope is retained.

Example 17: Determination of competition with other anti-FGFR2 antibodies

To analyze the competition between the anti-FGFR2 antibodies according to the invention and the anti-FGFR2 antibodies described in the art, different antibodies were evaluated in a competitive ELISA format: 20 μl of coating buffer at 4 ° C (# 121125 Candor) 2 μg/ml anti-human IgG (Fc specific (#I2136; sigma)) in Bioscience GmbH) was coated with an MTP disk (384 well Maxisorp, Nunc) overnight. After a washing step using 50 μl PBST (phosphate buffered saline, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, 0.05% Tween 20), 50 μl at 20 to 22 ° C 100% Smart Block (#113500, Candor Bioscience GmbH) blocked the plate for 1 hour and repeated the washing step 3 times. Incubate 20 μl for 1 hour at 20 to 22 ° C to 10% in PBST M048-D01-hIgG was fixed at a concentration of 1 μg/ml in the Smart Block (shown in Table 12, and the second row had M048-D01-hIgG1 capture); the control well without M048-D01-hIgG was only 10 in PBST % Smart Block incubation (shown in Table 12, row 2 with M048-D01-hIgG1 capture no). The fixation step was followed by three wash steps using 50 μl PBST. Pre-incubation consisting of recombinant human FGFR2, 10 nM (#10824-H08H, SinoBiological) and 5-fold serial dilution (1000 to 0.064 nM) of anti-FGFR2 IgG in PBST 10% SmartBlock (1 hour at A 20 μl quadruple of the antigen/antibody mixture at 20-22 ° C and incubated for 1 hour at 20 to 22 ° C, followed by three wash steps.

To detect recombinant FGFR2 protein, anti-His/HRP conjugate (#71840, novagen) diluted 1:10000 in PBST in 10% SmartBlock was administered at 20 to 22 °C for 1 hour. After 3 washing steps, add 20 μl of 10 μM amplex red substrate (# A12222, Invitrogen) in 50 mM sodium hydrogen phosphate, pH 7.6 and detect the fire using a common fluorescence reader (eg Tecan M1000) Optical signal.

Three different FGFR2 binding antibodies, described as GAL-FR21, GAL-FR22, and GAL-FR23 (described in WO2010/054265 and Zhao et al. (Clin Cancer Res. 2010, 16: 5750-5758) have been described to bind To different domain epitopes. To assess the differences in the use of these antibodies, a competitive assay was performed.

Because the antibodies to be analyzed have different isotypes, the competitive ELISA format must ensure equilibrium without direct overlap and direct detection of competitive status, since different detection antibodies or single detection antibodies are used differently. IgG-isotypes have different affinities. The above ELISA format satisfies this condition by detecting the FGFR2 antigen by detecting its binding to mouse or human IgG1 or IgG2a by His-tag substitution. Immobilization of M048-D01-hIgG1 is specific for its human Fc portion, otherwise a significant amount of FGFR2 will be in the ELISA wells coated with anti-human IgG (Fc specific) but not providing M048-D01-hIgG1 It was detected that mouse anti-FGFR2-IgG may bind to anti-human IgG (Fc specific) and thus no FGFR2 binding was detected (Table 12, columns 8-11). In addition, no significant non-specific binding of FGFR2 to immobilized anti-human IgG (Fc specific) was observed (column 2). The "self-competition" of "M048-D01-hIgG1" is very obvious (6 columns), and M048-D01-mIgG2a is also (column 7). None of GAL-FR21, -FR22 or -FR23 showed a dose-dependent decrease in detectable FGFR2 (columns 3-5), while M048-D01-hIgG1 and M048-D01-mIgG2a were at 1.25 and 0.63 nM and higher, respectively. The competitive antibody has a >50% down signal, which confirms the difference between M048-D01 and the three GAL antibodies. In contrast, after monomeric FGFR2 (10 nM) and GAL-FR22 and GAL-FR23, but not GAL-FR21, the number of detectable FGFR2 appeared to increase significantly. Because GAL antibodies are not fused to the His-tag but are detected by Western analysis and are not captured by anti-human IgG (Fc-specific), the most likely explanation is that monomeric FGFR2 may be pre-exposed with antibodies. Incubation and dimerization, while FGFR2 binds to the immobilized M048-D01-hIgG1 to produce a binding effect. The case where the immobilized M048-D01-hIgG1 is directly bound to FGFR2 and thereby indirectly mediated to the dimerized antibodies GAL-FR22 and GAL-FR23 will be further described. It is clear that M048-D01-hIgG1 binds to a completely different FGFR2 epitope than the GAL antibody, otherwise the binding event does not occur. It should be noted that GAL-FR21 does not increase the number of detectable FGFR2. This variability seems to be explained by taking into account a more specific description of the GAL antibody as described in WO2010/054265: Gal-FR22 binds to one epitope of D2-D3IIIa, whereas GAL-FR23 binds to all or part of it One of D1; two regions are indicated in the recombinant human FGFR2-IIIc molecule used. However, in the case of GAL-FR21, the epitope is described as a sequence segment in D3-IIIb that is not represented by this FGFR2-IIIc isoform: thus GAL-FR21 is unable to bind antigen and mediate binding effects. As shown, none of the analyses observed a competition between M048-D01-hIgG1 and one of the GAL antibodies.

The results of the competition experiments, as described above, are supported by the observation that all three GAL antibodies, including GAL-FR23 (all or part of the epitope in D1), show no extracellular N-terminus binding to FGFR2 a synthetic peptide of the epitope (SEQ ID NO: 63) comprising an amino acid sequence ( ¹ RPSFSLVEDTTLEPE ¹⁵ G-Ttds-Lys (biotin)) linked to the biotinylated lysine at the C-terminus of the epitope, Even at the highest concentration of IgG continuous titration (600 nM), M048-D01-hIgG1 (detected by anti-human IgG (Fc specific) POD conjugates: #A5175, sigma) and M048-D01-mIgG2a The strong combination makes the EC50 in the range of ≦1 nM (detailed data not shown). For the detection of mouse anti-system, anti-mouse IgG (Fc-specific) POD conjugate (#715-35-15, jackson) was used, and its ability was positively checked to detect GAL-binding to FGFR2-IIIbα. FR21, -FR22, -FR23 and M048-D01-mIgG2a.

Figure 1: Schematic diagram of the structure of FGFR2. The alpha (SEQ ID NO: 61) and beta (SEQ ID NO: 62) splice variants are shown in an aligned manner. This figure shows three Ig-like domains (D1, D2, and D3), transmembrane domain TM, and an intracellular kinase domain. Represents heparin binding site (HBS), acid box (AB) and alternation IIIb/IIIc local domain. The amine end is labeled N and the carboxy end is designated C. The binding epitope of the antibodies of the invention is shown as streaks.

Panel 2: Phosphorylated FGFR2 (P-FGFR2) concentration induced after short-term (15 minutes) incubation with 10 μg/ml of anti-FGFR2 antibody in MFM223 cells. Y is "% of untreated control cells". As shown, the ELISA signals of the antibodies M048-D01-hIgG1 and M047-D08-hIgG1 increasing P-FGFR2 were four times higher than the untreated control cells. In contrast, control IgG antibodies or anti-FGFR2 antibodies (MAB665, MAB684, MAB6843) commercially available from R&D did not show any significant effect on P-FGFR2 concentrations after short-term incubation. These results revealed the efficacious effect of the anti-FGFR2 antibody described in the present invention on FGFR2 after short-term incubation.

Figure 3: MFM223 cells were desensitized to FGF7 (25 ng/ml, 15 min) vehicle-induced P-FGFR2 concentration after long-term (24 h) incubation with 10 μg/ml anti-FGFR2 antibody. Y is "% of untreated control cells". As shown, antibodies M048-D01-hIgG1 and M047-D08-hIgG1 reduced the concentration of P-FGFR2, which was very evident after FGF7 stimulation. The concentration of FGF7 stimulated P-FGFR2 was increased about 4-fold in cells without antibody treatment and in cells treated with isoform control IgG. In contrast, FGF7 induced only a P-FGFR2 concentration of 1.37 to 1.4 fold in samples pretreated with anti-FGFR2 antibody for 24 hours. In summary, these results show that prolonged incubation of cells with the anti-FGFR2 antibodies of the invention would desensitize the stimulation with FGF7.

Figure 4: Overexpression with FGFR2 by FACS analysis 4.5 hours after incubation with 10 μg/ml of anti-FGFR2 antibody Down-regulation of FGFR2 surface expression in cell lines (MFM223, SNU16) or FGFR2 mutations (AN3-CA, MFE-296). Y is "control cell %". As shown, the antibodies M048-D01-hIgG1 and M047-D08-hIgG1 are FGFR2 surfaces which reduce the cell line with FGFR2 overexpression (MFM223, SNU16) and the cell line with FGFR2 mutation (AN3-CA, MFE-296). The only antibody that is expressed. Antibodies such as MAB684 and MAB6843 (R&D) only reduced the FGFR2 surface appearance of cell lines that did not overexpress FGFR2. Antibodies such as GAL-FR21 did not reduce the surface appearance of FGFR2 in cell lines with FGFR2 mutations.

Figure 5: The total FGFR2 concentration in SNU16 cells was down-regulated after long-term (96 hours) incubation with anti-FGFR2 antibodies. Y is "control cell %". X is "antibody concentration [μg/ml]". As shown, the antibodies M048-D01-hIgG1 (white) and M047-D08-hIgG1 (stripes) significantly reduced the total FGFR2 concentration after 96 hours in a dose-dependent manner. The non-binding control antibody (black) did not show any utility. These results indicate that the anti-FGFR2 antibodies M048-D01-hIgG1 and M047-D08-hIgG1 not only cause a short-term decrease in the surface FGFR2 concentration, but also a long-term decrease in the total FGFR2 concentration.

Figure 6: Microscopic evaluation of the time course of specific internalization of M048-D01-hIgG1 and M047-D08-hIgG1 after binding to cells expressing endogenous FGFR2. Y line "particle count per cell". X series "time [minutes]". Antibody internalization was studied on the breast cancer cell line SUM 52PE. The particle count for each cell is measured in a kinetic manner. As shown, the antibody M048-D01-hIgG1 (black squares and solid lines) and M047-D08-hIgG1 (black triangles and dashed lines) showed rapid internalization as shown by the increase in particle count per cell. Isotype control antibodies (star and dashed) did not show any internalization.

Figure 7: Internalization of M048-D01-hIgG1 (A, B) and M047-D08-hIgG1 (C, D) showed co-staining in SUM 52PE cells, such as using Rab 7 (A, C) instead of Rab 11 (B, D) is shown. Internalization of GAL-FR21 (E, F) and GAL-FR22 (G, H) showed co-staining in SUM 52PE cells, as shown using Rab 11 (F, H) instead of Rab 7 (E, G) .

Figure 8: Subcutaneous SNU-16 allograft at 2 mg/kg of M017-B02-hIgG1 (open triangle, compared to PBS (filled circles, solid line) and control IgG treatment (solid triangle, solid line) Solid line) Growth under intraperitoneal treatment. The average + standard deviation is plotted. X is "time after tumor inoculation [days]". Y is "tumor area [mm ² ]". Treatment with M017-B02-hIgG1 caused very significant inhibition of tumor growth.

Figure 9: Subcutaneous SNU-16 allograft at 2 mg/kg of M021-H02-hIgG1 (open triangle, compared to PBS (filled circles, solid line) and control IgG treatment (solid triangle, solid line) Solid line) Growth under intraperitoneal treatment. The average + standard deviation is plotted. X is "time after tumor inoculation [days]". Y is "tumor area [mm ² ]". Treatment with M021-H02-hIgG1 caused very significant inhibition of tumor growth.

Figure 10: Subcutaneous SNU-16 allograft at 2 mg/kg of M048-D01-hIgG1 (open triangle, compared to PBS (filled circles, solid line) and control IgG treatment (solid triangle, solid line) Solid line) Growth under intraperitoneal treatment. The average + standard deviation is plotted. X is "time after tumor inoculation [days]". Y is "tumor area [mm ² ]". Treatment with M048-D01-hIgG1 caused very significant inhibition of tumor growth.

Figure 11: Subcutaneous SNU-16 allograft at 2 mg/kg of M054-A05-hIgG1 (open triangle, compared to PBS (filled circles, solid line) and control IgG treatment (solid triangle, solid line) Solid line) Growth under intraperitoneal treatment. The average + standard deviation is plotted. X is "time after tumor inoculation [days]". Y is "tumor area [mm ² ]". Treatment with M054-A05-hIgG1 caused very significant inhibition of tumor growth.

Figure 12: Growth of subcutaneous SNU-16 allografts under intraperitoneal treatment with 2 mg/kg of M054-D03-hIgG1 (open triangle, solid line) compared to PBS (filled circles, solid line). The average + standard deviation is plotted. X is "time after tumor inoculation [days]". Y is "tumor area [mm ² ]". Treatment with M054-D03-hIgG1 caused very significant inhibition of tumor growth.

Figure 13: Growth of subcutaneous SNU-16 allografts under intraperitoneal treatment with 2 mg/kg of M047-D08-hIgGl (open triangle, solid line) compared to PBS (filled circles, solid line). The average + standard deviation is plotted. X is "time after tumor inoculation [days]". Y is "tumor area [mm ² ]". Treatment with M047-D08-hIgG1 caused very significant inhibition of tumor growth.

Figure 14: Dot pattern of tumor area of subcutaneous 4T1 tumor on day 13 after tumor cell inoculation, the tumor became necrotic before the last time point. Mice received either PBS (A), 5 mg/kg M048-D01-hIgG1 twice weekly iv (B), 100 mg/kg lapatinib po (C) or 5 mg/ at this time point. Kg of M048-D01-hIgG1 was treated twice weekly with iv and 100 mg/kg lapatinib po(D). Y is the tumor area [mm ² ] on the 13th day, the broken line indicates the average value, and the solid line indicates the median. Treatment with M048-D01-hIgG1 alone resulted in a significant reduction in tumor area, whereas lapatinib alone did not significantly affect tumor area. The combination of M048-D01-hIgG1 with lapatinib resulted in significant additive anti-tumor activity.

Figure 15: A dot plot of the tumor area of a subcutaneous 4T1 tumor on day 13 after tumor cell inoculation, with the tumor becoming necrotic until the last time point. Mice received PBS (A) at this time point, 5 mg/kg of M048-D01-hIgG1 twice weekly iv (B), 24 mg/kg paclitaxel once a week iv (C) or 5 mg /kg of M048-D01-hIgG1 twice weekly iv and 24 mg/kg paclitaxel once weekly iv (D) treatment. Y is the tumor area [mm ² ] on the 13th day, the broken line indicates the average value, and the solid line indicates the median. Treatment with M048-D01-hIgG1 alone resulted in a significant reduction in tumor area, whereas paclitaxel alone did not significantly affect tumor area. The combination of M048-D01-hIgG1 with paclitaxel resulted in significant additive anti-tumor activity.

Figure 16: Compared to PBS (open diamond, solid line), the patient derived subcutaneous GC10-0608 allograft at 5 mg/kg (solid triangle, solid line), 2 mg/kg (closed round) , dotted line) growth with M048-D01-hIgG1 treated with 1 mg/kg (closed square, dotted line). The average + standard error is plotted. X is "processing time [day]". Y is "tumor volume [mm ³ ]". Treatment with all three doses of M048-D01-hIgG1 resulted in significant tumor growth inhibition.

Figure 17: Patient-derived subcutaneous GC12-0811 allograft at 5 mg/kg (solid triangle, solid line), 2 mg/kg (closed circle) compared to PBS (open diamond, solid line) , dotted line) growth with M048-D01-hIgG1 treated with 1 mg/kg (closed square, dotted line). The average + standard error is plotted. X is "processing time [day]". Y is "tumor volume [mm ³ ]". Treatment with M048-D01-hIgG1 at doses of 5 and 1 mg/kg resulted in significant tumor growth inhibition.

Figure 18: Long-term anti-FGFR2 antibody M048-D01-hIgG1 and M047-D08-hIgG1 compared to control antibody (2 mg/kg twice a week, ip, sample obtained 24 hours after the last administration) Downregulation of total FGFR2 [total FGFR2] and phosphorylated FGFR2 [P-FGFR2] after treatment of SNU16 allografts. As shown, total FGFR2 [total FGFR2] was significantly reduced compared to phosphorylated FGFR2 [P-FGFR2] compared to control IgGl after treatment with M048-D01-hIgG1 and M047-D08-hIgG1. Actin was used as a loading control.

Figure 19: Sequence of the invention.

<110> Bayer Intellectual Property GmbH

<120> Anti-FGFR2 antibody and use thereof

<130> BHC 11 1 042

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<211> 110

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<400> 94

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<211> 8

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<400> 175

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<211> 11

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Claims (25)

An isolated antibody or antigen-binding fragment thereof, which is in a cell line SNU16 (ATCC-CRL-5974) and MFM223 (ECACC-98050130) which overexpress FGFR2 and a cell line AN3-CA (DSMZ-ACC) which exhibits mutant FGFR2 267) Reduced cell surface expression of FGFR2 after binding to FGFR2 with MFE-296 (ECACC-98031101). An isolated antibody or antigen-binding fragment thereof that specifically binds to the extracellular N-terminal epitope of FGFR2 as set forth in SEQ ID NO: 63 ( ¹ RPSFSLVEDTTLEPE ¹⁵ ). An isolated antibody or antigen-binding fragment thereof according to claim 2, wherein the antibody binds to an extracellular N-terminal epitope (SEQ ID NO: 63) by at least one selected from the group consisting of the following residues The medium of the epitope residues: Arg 1, Pro 2, Phe 4, Ser 5, Leu 6 and Glu 8. The isolated antibody or antigen-binding fragment according to any one of claims 2 to 3, wherein the antibody or antigen-binding fragment thereof is at least one of an N-terminal epitope ( ¹ RPSFSLVEDTTLEPE ¹⁵ ) of FGFR2 The amino acid residue is changed to alanine and loses more than 50% of the ELISA signal, a) the residue is selected from the group of Pro 2, Leu 6 and Glu 8 or b) the residue is selected from Arg 1 Pro 2, Phe 4 and Ser 5 groups. An antibody or antigen-knot as claimed in any one of claims 1 to 4 a fragment, wherein the antibody or antigen-binding fragment competes for binding to at least one antibody selected from the group consisting of FGFR2: "M048-D01", "M047-D08", "M017-B02", "M021-H02", M054-A05, M054-D03, TPP-1397, TPP-1398, TPP-1399, TPP-1400, TPP-1401, TPP-1402, TPP -1403", "TPP-1406", "TPP-1407", "TPP-1408", "TPP-1409", "TPP-1410", "TPP-1411", "TPP-1412" and "TPP-1415" ". The antibody or antigen-binding fragment according to any one of claims 5, wherein the amino acid sequence of the antibody or antigen-binding fragment is at least 50%, 55%, 60% with at least one of the following CDR sequences, 70%, 80%, 90 or 95% identical: "M048-D01", "M047-D08", "M017-B02", "M021-H02", "M054-A05", "M054-D03", "TPP -1397", "TPP-1398", "TPP-1399", "TPP-1400", "TPP-1401", "TPP-1402", "TPP-1403", "TPP-1406", "TPP-1407" "," TPP-1408", "TPP-1409", "TPP-1410", "TPP-1411", "TPP-1412" or "TPP-1415", or at least 50% with the following VH or VL sequences, 60%, 70%, 80%, 90%, 92% or 95% identical: "M048-D01", "M047-D08", "M017-B02", "M021-H02", "M054-A05"," M054-D03", "TPP-1397", "TPP-1398", "TPP-1399", "TPP-1400", "TPP-1401", "TPP-1402", "TPP-1403", "TPP-" 1406”, “TPP-1407”, “TPP-1408”, “TPP-1409”, “TPP-1410”, “TPP-1411”, “TPP-1412” or “ TPP-1415”. The antibody or antigen-binding fragment according to any one of claims 5 to 6, wherein the antibody or antigen-binding fragment comprises at least one CDR sequence as shown in Table 9 and Table 10 or at least one variable weight Chain or light chain sequence. An antibody or antigen-binding fragment according to claims 1 to 7 which comprises: a variable heavy CDR sequence as represented by SEQ ID NOs: 5-7 and represented by SEQ ID NOs: 8-10 Variable light chain CDR sequences, or b such as the variable heavy chain CDR sequences represented by SEQ ID NOs: 15-17 and the variable light chain CDR sequences represented by SEQ ID NOs: 18-20, or c as SEQ ID The variable heavy chain CDR sequences represented by NO: 25-27 and the variable light chain CDR sequences represented by SEQ ID NOs: 28-30, or d the variable heavy chains represented by SEQ ID NOs: 35-37 CDR sequences and variable light chain CDR sequences represented by SEQ ID NOS: 38-40, or e variable heavy chain CDR sequences as set forth in SEQ ID NOs: 45-47 and by SEQ ID NO: 48-50 a variable light chain CDR sequence represented, or a variable heavy CDR sequence as represented by SEQ ID NOs: 55-57, and a variable light chain CDR sequence represented by SEQ ID NOs: 58-60, or g such as the variable heavy CDR sequences represented by SEQ ID NOS: 75-77 and the variable light CDR sequences represented by SEQ ID NOS: 78-80, or h as represented by SEQ ID NOs: 85-87 The variable heavy chain CDR sequences and the variable light chain CDR sequences represented by SEQ ID NOS: 88-90, or the variable heavy CDR sequences represented by SEQ ID NOs: 95-97, and SEQ ID NO: The variable light chain CDR sequences represented by 98-100, or the variable heavy chain CDR sequences represented by SEQ ID NOs: 105-107 and the variable light chain CDR sequences represented by SEQ ID NOs: 108-110 Or k as the variable heavy CDR sequences represented by SEQ ID NOS: 115-117 and the variable light CDR sequences represented by SEQ ID NOS: 118-120, or 1 as SEQ ID NO: 125-127 The indicated variable heavy chain CDR sequences and the variable light chain CDR sequences represented by SEQ ID NOs: 128-130, or m the variable heavy chain CDR sequences represented by SEQ ID NOs: 135-137 and by SEQ ID NO: a variable light chain CDR sequence represented by 138-140, or n a variable heavy CDR sequence represented by SEQ ID NO: 145-147 and a variable light chain represented by SEQ ID NO: 148-150 CDR sequence, or o a variable heavy CDR sequence as represented by SEQ ID NO: 155-157 and a variable light chain CDR sequence represented by SEQ ID NO: 158-160, or p as represented by SEQ ID NO: 165-167 The variable heavy chain CDR sequences and the variable light chain CDR sequences represented by SEQ ID NOs: 168-170, or the variable heavy chain CDR sequences represented by SEQ ID NOs: 175-177, and SEQ ID NO: The variable light chain CDR sequences represented by 178-180, or the variable heavy chain CDR sequences represented by SEQ ID NOs: 185-187, and the variable light chain CDR sequences represented by SEQ ID NOs: 188-190 Or s a variable heavy CDR sequence as represented by SEQ ID NO: 195-197 and a variable light CDR sequence represented by SEQ ID NO: 198-200, or t as set forth in SEQ ID NO: 205-207 The indicated variable heavy chain CDR sequences and the variable light chain CDR sequences represented by SEQ ID NOs: 208-210, or the variable heavy CDR sequences represented by SEQ ID NOs: 215-217, and by SEQ ID NO: The variable light chain CDR sequence represented by 218-220. An antibody or antigen-binding fragment according to claims 1-8, which comprises: a variable heavy chain sequence as represented by SEQ ID NO: 1 and a variable light chain sequence as represented by SEQ ID NO: 2, or b as a variable heavy chain sequence represented by SEQ ID NO: 11 and a variable light chain sequence as represented by SEQ ID NO: 12, or c as The variable heavy chain sequence represented by SEQ ID NO: 21 and the variable light chain sequence represented by SEQ ID NO: 22, or d the variable heavy chain sequence represented by SEQ ID NO: 31 and SEQ ID NO: The variable light chain sequence represented by 32, or the variable heavy chain sequence represented by SEQ ID NO: 41 and the variable light chain sequence as represented by SEQ ID NO: 42, or f such as SEQ ID NO: The variable heavy chain sequence represented by 51 and the variable light chain sequence represented by SEQ ID NO: 52, or the variable heavy chain sequence represented by SEQ ID NO: 73 and represented by SEQ ID NO: 74 a variable light chain sequence, or h such as the variable heavy chain sequence represented by SEQ ID NO: 83 and the variable light chain sequence set forth in SEQ ID NO: 84, or i as represented by SEQ ID NO: 93 A variable heavy chain sequence and a variable light chain sequence as represented by SEQ ID NO: 94, or j a variable heavy chain sequence as represented by SEQ ID NO: 103 and a variable as represented by SEQ ID NO: 104 a strand sequence, or k a variable heavy chain sequence as represented by SEQ ID NO: 113 and a variable light chain sequence as represented by SEQ ID NO: 114, or a variable heavy chain as represented by SEQ ID NO: 123 Sequence And a variable light chain sequence as represented by SEQ ID NO: 124, or a variable heavy chain sequence represented by SEQ ID NO: 133 and a variable light chain sequence as represented by SEQ ID NO: 134, or n a variable heavy chain sequence as represented by SEQ ID NO: 143 and a variable light chain sequence as represented by SEQ ID NO: 144, or o a variable heavy chain sequence as represented by SEQ ID NO: 153 and SEQ ID NO: The variable light chain sequence represented by 154, or p such as the variable heavy chain sequence represented by SEQ ID NO: 163 and the variable light chain sequence as represented by SEQ ID NO: 164, or q such as SEQ ID NO a variable heavy chain sequence represented by :173 and a variable light chain sequence as represented by SEQ ID NO: 174, or a variable heavy chain sequence represented by SEQ ID NO: 183 and as set forth in SEQ ID NO: 184 a variable light chain sequence represented, or a variable heavy chain sequence as represented by SEQ ID NO: 193 and a variable light chain sequence as represented by SEQ ID NO: 194, or t as represented by SEQ ID NO: 203 Variable heavy chain sequence And a variable light chain sequence as represented by SEQ ID NO: 204, or a variable heavy chain sequence represented by SEQ ID NO: 213 and a variable light chain sequence as set forth in SEQ ID NO: 214. An antibody according to any one of the preceding claims, which is an IgG antibody. An antigen-binding fragment according to any one of the preceding claims, which is a scFv, Fab, Fab' fragment or F(ab') ₂ fragment. An antibody or antigen-binding fragment according to any one of the preceding claims, which is a monoclonal antibody or an antigen-binding fragment. An antibody or antigen-binding fragment according to any one of the preceding claims, which is a human, humanized or chimeric antibody or antigen-binding fragment. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof according to claims 1 to 13 of the patent application. An isolated nucleic acid sequence encoding an antibody or antigen-binding fragment as claimed in claims 1 to 13. A vector comprising the nucleic acid sequence of claim 15 of the patent application. An isolated cell which exhibits an antibody or antigen-binding fragment according to any one of claims 1 to 13 and/or a nucleic acid according to item 15 of the patent application or a carrier of claim 16 of the patent application. The isolated cell of claim 17, wherein the cell is a prokaryotic cell or a eukaryotic cell. A method of producing an antibody or antigen-binding fragment according to any one of claims 1 to 13, which comprises culturing a cell as claimed in claim 18 and purifying the antibody or antigen-binding fragment. An antibody or antigen-binding fragment according to claims 1 to 13 or an antibody-drug conjugate according to claim 14 of the patent application, which is a drug. An antibody or antigen-antigen-binding fragment as claimed in claims 1 to 13 is used as a diagnostic agent. An antibody or antigen-binding fragment according to claims 1 to 13 or an antibody-drug conjugate according to claim 14 of the patent application, which is a medicament for treating cancer. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to claims 1 to 13 of the patent application or an antibody-drug conjugate according to claim 14 of the patent application. A composition of a pharmaceutical composition according to claim 23 of the patent application and one or more therapeutically active compounds. A method of treating a condition or condition associated with an undesired presence of FGFR2, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition as disclosed in claim 23 or a composition as in claim 24 of the patent application.